Bifunctional chelating agents

ABSTRACT

The present invention provides bifunctional chelating agents comprising a unique substrate reactive moiety incorporated into a carboxymethyl arm of a polyaminopolycarboxylate chelating framework capable of forming stable complexes with metal ions.

This is a division of pending prior application Ser. No. 07/136,180,filed Jan. 4, 1988, which is a continuation in part of 07/014,517, filedon Feb. 13, 1987. Patent application Ser. No. 07/014,517 has beenabandoned in favor of Ser. No. 07/136,180, now U.S. Pat. No. 5,057,302.

BACKGROUND

The present invention relates generally to chelating agents for thebinding of metal ions to biologically active molecules, both naturallyoccurring and synthetic. Specifically, it relates to bifunctionalchelating agents comprising an array of metal binding groups plus asingle additional moiety (hereafter a "substrate reactive" group) whichis reactive with one or more functionalities present on the molecule tobe labelled with a metal ion (hereafter the "substrate"). According toone embodiment, the invention relates to antibody conjugates andantibody metal ion conjugates comprising the bifunctional chelatingagents and to the use of such antibody-metal ion conjugates for in vivodiagnostic imaging methods utilizing radiation emitting and radiationabsorbing metal ions. The invention additionally relates to therapeuticmethods involving the use of such antibody-metal ion conjugates whichemit cytotoxic radiation.

Of interest to the present invention are disclosures showing the use ofprotein/metal ion conjugates for diagnostic and therapeutic purposes.Gansow, et al., U.S. Pat. No. 4,454,106 discloses the use of monoclonalantibody/metal ion conjugates for in vivo and in vitro radioimagingdiagnostic methods. Goldenberg, et al., N. Eng. J. Med., 298 1384-88(1978) discloses diagnostic imaging experiments wherein antibodies tothe known tumor associated antigen carcinoembryonic antigen (CEA) arelabelled with iodine¹³¹ and injected into patients with cancer. After 48hours, the patients are scanned with a gamma scintillation camera andtumors are localized by the gamma emission pattern.

Other workers disclose the therapeutic use of antibody/metal ionconjugates for delivery of cytotoxic radioisotopes to tumor deposits invivo. Order, et al., Int. J. Radiation Oncology Biol. Phys., 12, 277-81(1986) describes treatment of hepatocellular cancer with antiferritinpolyclonal antibodies to which yttrium⁹⁰ has been chelated. Duchsbaum,et al., Int. J. Radiation Oncology Biol. Phys., 12, 79-82 (1985)discloses radiolabelling of monoclonal antibodies to CEA with yttrium⁸⁸and suggests the possibility of localization and treatment of colorectalcancers therewith. Nicolotti, EPO Application No. 174,853 published Mar.19, 1986, discloses conjugates comprising metal ions and antibodyfragments. According to that disclosure, monoclonal antibodies ofsubclass IgG are enzymatically treated to remove the Fc fragment andreductively cleave the disulfide bond linking the antibody heavy chains.The Fab' fragment is then linked to a chelating agent bound to aradionuclide metal ion for in vivo diagnostic or therapeutic use.

Antibody/metal ion conjugates may be formed through the use ofbifunctional chelating agents comprising an array of metal-bindinggroups plus a moiety capable of covalent binding to a protein substrate.Early work with bifunctional chelating agents involved the compounddiethylenetriaminepentaacetic acid (DTPA) and its derivatives. Thiscompound comprises a backbone of three nitrogen atoms linked by twoethylene chains. Extending from the nitrogen atoms on the backbone arefive carboxymethyl moieties. Methods have been described whereby one ofthe carboxymethyl groups may be reacted to form an amide bond with anamino acid residue present on an antibody or other protein molecule. Theother four carboxymethyl moieties, together with the three nitrogenatoms, then remain available for metal binding. Unfortunately, becausethere is no intrinsic difference between the substrate reactive andchelating functionalities in DTPA, such procedures can lead to crosslinking and denaturation of the antibody with concomitant degradation ofits ability to bind to the target antigen.

In order to avoid the potential for such undesired cross-linking,bifunctional chelating agents incorporating a unique protein substratereactive site have been developed. The first such compounds werederivatives of the compound ethylenediaminetetraacetic acid (EDTA). Thiscompound comprises a backbone of two nitrogen atoms linked by anethylene chain. Extending from the nitrogen atoms on the backbone arefour carboxymethyl moieties which with the nitrogen atoms are suitablefor metal binding. The bifunctional chelating derivatives of EDTA arecharacterized by the attachment of a unique protein substrate reactivefunction at a methylene carbon of the polyamine backbone. Sundberg, etal., J. Med. Chem. 17, 1304 (1974) discloses the synthesis of an EDTAderivative bearing a para aminophenyl protein reactive substituent. Thisderivative may in turn be converted to bifunctional chelating agentscapable of being coupled to protein substrates under mild conditionseither by reaction of the amine with a portion of a chemically modifiedprotein or by treatment of the primary amine to form other substituentscapable of binding to protein substrates under mild conditions.

Meares, et al., J. Protein Chem., 3, 215-228 (1984) discloses methodswhereby the para aminophenyl derivative is converted to a diazoniumderivative through nitrous acid treatment, to an isothiocyanatederivated by treatment with thiophosgene, to a bromoacetamide derivativeby treatment with bromoacetylbromide and to a palmitaamidobenzylderivative by treatment with palmitoyl chloride. Altman, et. al., J.Chem. Soc. Perkin Trans. I., 365, 59-62 (1983) discloses a number ofphenethyl analogues of the above EDTA compounds. See also, Sundberg, etal., U.S. Pat. No. 3,994,966.

Cyclic chelating agents are known in the art. Kroll, et al., Nature, 180919-20 (1957) discloses the use ofcyclohexane-1,2-trans-diaminetetraacetic acid for the removal of heavymetal ions from the human body. Moi, et al., Anal. Biochem., 148,249-253 (1985) discloses a macrocyclic bifunctional chelating agentprecursor named 6-(p-nitrobenzyl)-1,4,8,11-tetra-azacyclotetradecaneN,N',N'', N'''-tetraacetic acid (p-nitrobenzyl-TETA) which forms acopper chelate which is extremely stable in human serum underphysiological conditions. In addition, the p-bromoacetamidobenzylderivative of TETA shows high stability after conjugation to amonoclonal antibody. The Moi, et al. reference also discloses thatimproved metal binding yields may be obtained in some cases where theconjugate contains a spacer group between the protein and TETA.

Of interest to the present application are the disclosures of Green, etal., Int. J. Nucl. Med. Biol., 12, 381-85 (1985) and Taliaferro, et al.,Inorg. Chem. 23 1188-92 (1984) disclosing chelating agents. Green, etal. discloses a sexadentate ligandN,N'-dipyridoxylethylenediamine-N,N'-diacetic acid (PLED) complexed withgallium⁶⁸ and indium¹¹¹. Taliaferro, et al., discloses PLED chelates aswell as those of N,N'-ethylene-bis[2-(o-hydroxy phenyl)glycine] (EHPG)and N,N'-bis(2- hydroxybenzyl) ethylenediamine ,N,N'-diacetic acid(HBED).

Other variations on known DTPA and EDTA derivatives include those ofBrechbiel, et al., Inorg. Chem., 25, 2772-81 (1986) which disclosesderivatives of DTPA wherein para-aminophenyl substituents are attachedto the methylene carbons of the polyamine backbone. In addition, Altman,et al., J. Chem. Soc. Perkin Trans. I., 59 (1984) discloses a2-carboxyethyl chelating derivative of EDTA.

Methods of synthesizing derivatives of DTPA and EDTA wherein the proteinreactive functionality is attached to a methylene carbon of thepolyamine backbone are complex, difficult to practice and have limitedflexibility. In addition, the polyamine backbone carbon atoms of manychelating agents such as those wherein such carbons are part of a cyclicsystem are not readily available for substitution. It is thereforedesired to develop bifunctional chelating agents having a uniquesubstrate reactive function attached to a moiety common to, andaccessible in, all polyaminopolycarboxylate frameworks. It is furtherdesired to develop general methods for the synthesis of such compounds.

Of interest to the present invention is the disclosure of Takeshita andMaeda, Yukagaku, 19, 984-93 (1970) which shows a surfactant compoundwith the structure: ##STR1##

wherein R is a long chain alkyl.

Also of interest to the present invention is the disclosure of Borch, etal., J. Amer. Chem. Soc., 93 2397 (1971) showing the reductive aminationof a variety of pyruvic acids including phenylpyruvic andpara-hydroxyphenylpyruvic acid to the corresponding dl-alpha amino acidsunder mild conditions using ammonia and sodium cyanoborohydride.

SUMMARY OF THE INVENTION

The present invention provides bifunctional chelating agents comprisinga unique "substrate reactive" moiety incorporated into a carboxymethylarm of a polyaminopolycarboxylate chelating framework capable of formingstable complexes with metal ions. Suitable substrate reactive groupsinclude phenyl groups directly substituted or substituted throughaliphatic spacer arms with substrate reactive moieties such as amino,thiocyanato, diazonium and bromoacetamide which are capable of reactingwith one or more functionalities present on the substrate molecule to belabelled with a metal ion. The substrate reactive groups also comprisephenyl groups directly substituted or substituted through aliphaticspacer groups with substrate reactive moieties such as thiosemicarbazideand hydrazine.

The present invention is particularly advantageous in that it provides ageneral method for the introduction of a versatile substrate reactivemoiety into any polyaminopolycarboxylic acid structure withoutcompromising the metal binding properties of that structure. The priorart synthetic methods, whereby a substrate reactive moiety is introducedat a methylene carbon atom of the polyamine backbone, cannot readily beextrapolated to the synthesis of many other desired chelating agentssuch as those wherein these ethylene carbon atoms are part of a ringsystem. By providing compounds in which the substrate reactive functionis attached at a moiety common to, and accessible in, allpolyaminopolycarboxylate frameworks, the present invention provides ameans for applying the full spectrum of chelating properties exhibitedby such frameworks to problems involving the labelling of biologicallyactive molecules with metal ions.

The bifunctional chelating agents of the invention are suitable forbinding metals including radioactive metal ions to a variety ofsubstrate molecules including, but not limited to proteins,glycoproteins, peptides, poly(amino acids), lipids, carbohydrates,polysaccharides, nucleosides, nucleotides, nucleic acids, bile acids,drugs, inhibitors and intact cells. Compounds according to the inventionhave as many as 10 or more metal binding substituents with the highersubstituted compounds having improved stability when chelating metalions with high coordination numbers such as lanthanide and actinidemetals. The invention also provides cyclic bifunctional chelator systemswhich provide modified stability properties.

According to one aspect of the invention, antibody conjugates andantibody-metal ion conjugates are provided comprising the bifunctionalchelating agents. In addition, the invention further provides in vivodiagnostic imaging methods utilizing radiation emitting and radiationabsorbing metal ions. According to a further aspect of the invention,therapeutic methods are provided for treatment of conditions such ascancer whereby cytotoxic radiation emitting nuclides are bound toanti-tumor specific antigen antibodies by the chelating agents of theinvention. The antibody radionuclide conjugates according to theinvention are then introduced into a subject and the cytotoxicradionuclides are selectively directed to cells bearing the tumorassociated antigen.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1a is a graph showing the results of an ELISA assay comparing thedegree of immune reactivity retained by an anti-CEA-DTTA(0.5) antibodyconjugate compared with unconjugated anti-CEA.

FIG. 1b is a graph showing the results of an ELISA assay comparing thedegree of immune reactivity retained by an anti-CEA-DTTA(5.0) antibodyconjugate compared with unconjugated anti CEA.

FIG. 1c is a graph showing the results of an ELISA assay comparing thedegree of immune reactivity retained by an anti-CEA-NCS-Compound Aantibody conjugate compared with unconjugated anti-CEA.

DETAILED DESCRIPTION

The present invention provides bifunctional chelating agents comprisinga unique substrate reactive moiety incorporated into a carboxymethyl armof a polycarboxylate chelating framework which is capable of formingstable complexes with metal ions. The compounds of the invention arespecifically characterized by having the structure: ##STR2## wherein Xis meta or para and is nitro or a substrate reactive moiety; wherein n=0to about 10;

wherein R₁ is selected from the group consisting of:

    --(CH.sub.2).sub.q,

    --[(CH.sub.2).sub.q N(R.sub.5)CH.sub.2).sub.r ],

    --[(CH.sub.2).sub.q O(CH.sub.2).sub.r O(CH.sub.2).sub.s ]--,

    --[(CH.sub.2).sub.q N(R.sub.5)(CH.sub.2 --.sub.r N(R.sub.6)(CH.sub.2).sub.s ]--, ##STR3##  and wherein

q=2 or 3,

r=2 or 3, and

s=2 or 3;

wherein R₂, R₃, R₅ and R₆ are the same or different and are selectedfrom the group consisting of:

hydrogen

CH₂ CO₂ H

ortho-CH₂ C₆ H₄ OH, and

wherein when R₁ =(CH₂)q

R₂ and R₃ may be fused to form a hydrocarbyl ring of the formula

    --(CH.sub.2).sub.t N(R.sub.7)(CH.sub.2).sub.u N(R.sub.8)(CH.sub.2).sub.v --

wherein

t=2 or 3,

u=2 or 3, and

v=2 or 3

wherein R₇ and R₈ are selected from the group consisting of:

hydrogen,

--CH₂ CO₂ H, and

ortho-CH₂ C₆ H₄ OH.

While the present invention provides numerous bifunctional chelatingagents comprising a variety of substrate reactive moieties and chelatingfunctionalities of various sizes, shapes and denticities, there is nosingle preferred chelating agent according to the invention. Preferredcompounds vary according to the specific nature of the substrate andmetal ion to be bound. Nevertheless, particular substituents andformations tend to be generally preferred under particularcircumstances.

When X is nitro, it is understood that further conversion to a substratereactive moiety is required prior to reaction with a substrate.

Preferred substrate reactive groups for X include those selected fromthe group consisting of:

    ______________________________________                                        NH.sub.2,           (AMINO)                                                   NN.sup.+,           (DIAZONIUM)                                               NCS,                (ISOTHIOCYANATE)                                          NCO,                (ISOCYANATE)                                              NHNH.sub.2          (HYDRAZINE)                                               NHCONHNH.sub.2,     (SEMICARBAZIDE)                                           NHCSNHNH.sub.2,     (THIOSEMI-                                                                    CARBAZIDE)                                                NHCOCH.sub.2 Cl,    (CHLORO-                                                                      ACETAMIDE)                                                NHCOCH.sub.2 Br,    (BROMOACETAMIDE)                                          NHCOCH.sub.2 I,     (IODOACETAMIDE)                                           N.sub.3,            (AZIDE)                                                   NHCONH(CH.sub.2).sub.m NH.sub.2,                                                                  (AMINOALKYLUREA)                                          NHCSNH(CH.sub.2).sub.m NH.sub.2,                                                                  (AMINOALKYL-                                                                  THIOUREA)                                                  ##STR4##           (MALEIMIDE)                                                ##STR5##           (HALOTRIAZINE)                                            and,                                                                           ##STR6##           (META-(DIHYDROXY- BORYL)PHENYL- THIOUREA)                 ______________________________________                                    

wherein Y is selected from the group consisting of Cl, Br and F;

wherein Z is selected from the group consisting of Cl, Br, F, OH andOCH₃ ;

wherein m=1 to about 10.

Particularly preferred substrate reactive moieties include those whereinX is para substituted and is selected from the group consisting of:--NH₂, --NCS, --NHCOCH₂ Br and --NHCSNH(CH₂)₂ NH₂. Where it is desiredto avoid direct linkage with the amino acid side chain of a proteinsubstrate, preferred substrate reactive moieties include those which arecapable of reaction with glycosylation present on some proteins. Becausesuch glycosylation is generally inert, it must often be derivatized oroxidized to increase its reactivity. Substrate reactive moietiespreferred for use in binding glycosylated proteins include --NHNH₂ and--NHCSNHNH₂.

Preferred substituents for R₁ include those selected from the groupconsisting of:

    --(CH.sub.2)--

    --[(CH.sub.2).sub.2 N(CH.sub.2 CO.sub.2 H)(CH.sub.2).sub.2 ]--

    --[(CH.sub.2).sub.2 O(CH.sub.2).sub.2 O(CH.sub.2).sub.2 ]--

    --[(CH.sub.2 (.sub.2 N)CH.sub.2 CO.sub.2 H)(CH.sub.2).sub.2 N(CH.sub.2 CO.sub.2 H)(CH.sub.2).sub.2 ]-- ##STR7## and

Substituents R₂, R₃, R₄, R₅, R₆, R₇ and R₈ are preferably selected fromthe group consisting of hydrogen and CH₂ CO₂ H. Preferred compounds forcertain applications include those wherein R₂ and R₃ are not fused toform a hydrocarbyl ring and wherein R₂, R₃, R₄, R₅ and R₆ are selectedfrom the group consisting of hydrogen and CH₂ CO₂ H. Particularlypreferred for certain applications are those compounds wherein R₂, R₃,R₄ and R₅ are CH₂ CO₂ H.

Preferred compounds further include those wherein n=1. Particularlypreferred for certain applications are chelating agents wherein R₁ is[CH₂ --CH₂ ]-- or --[(CH₂)₂ N(CH₂ CO₂ H)(CH₂)₂ ]-- and X is NCS. Aparticularly preferred group of compounds includes those wherein X ispara and is selected from the group consisting of --NO₂, --NH₂, --NCS,--NHCSNHNH₂ and --NHCSNH(CH₂)₂ NH₂ wherein n=1, wherein R₁ is selectedfrom the group consisting of

    [(CH.sub.2).sub.2 --,

    [(CH.sub.2).sub.2 O(CH.sub.2).sub.2 O(CH.sub.2).sub.2 ]--,

    --[(CH.sub.2).sub.2 N(CH.sub.2 CO.sub.2)(CH.sub.2 ]--and ##STR8## and wherein R.sub.2, R.sub.3, R.sub.4 are --CH.sub.2 CO.sub.2 H.

A preferred compound with a denticity of 3 is defined by the structure:##STR9##

Even while certain substituents and moieties of the chelating compoundsof the invention are generally preferred, the specific structures of thecompounds of the invention vary widely according to the natures of thesubstrate and the metal ion to be bound. Nevertheless, certain generalprinciples may be applied to tailoring the compounds of the invention tospecific uses. For example, certain metal ions such as members of theactinide and lanthanide series of metals have large radii and preferhigh coordination numbers. Preferred chelating agents for binding withsuch ions are larger compounds with increased denticity. As anotherexample, the precise location of particular amino acid residues in thestructure of a protein (on or near an active site of an enzyme, or on ornear the recognition region of an antibody), may require the selectionof an alternative substrate reactive moiety on a chelating agent. It mayalso occur that macrocyclic chelating agents according to the inventionmay provide improved stability characteristics for binding of certainmetal ions. Accordingly, multiple aspects of the present invention aresubject to variation.

Aliphatic chain lengths may be varied within the core structure anddenticity may be increased by the incorporation of metal reactivecarboxymethylamino groups on the R₁ chain. Chelating agents according tothe invention comprising increasing levels of denticity include N(carboxymethyl)-N-(2-(bis(carboxymethyl)amino)ethyl)-(4-aminophenyl)alanine (hereinafter "NH₂ -Compound A") witha denticity of 6, N-(carboxymethyl)-N (2 aminoethyl)N'-(carboxymethyl)-N'-(2'-(bis(carboxymethyl)amino)ethyl)-(4-aminophenyl)alanine(hereinafter "NH₂ -Compound B") with a denticity of 8, andN-(carboxymethyl)-N-(2-aminoethyl)-N'-(carboxymethyl)-N'-(2'-aminoethyl)-N''-(carboxy-methyl)-N''-(2''(bis(carboxymethyl)-amino)ethyl)-(4-aminophenyl)alaninewith a denticity of 10. ##STR10##

Such chelating agents with increasing denticities ranging from about 6to 10 or more are particularly useful for chelating metals which favorhigh coordination numbers such as members of the lanthanide and actinideseries.

The R₁ group may also be selected so as to introduce steric constraintsinto the diamine backbone by making the backbone part of a ring system.The introduction of such steric constraints in either non-aromatic oraromatic ring form leads to modified stability for certain metalcomplexes. Chelating agents according to the invention wherein thebackbone has been so modified include N-(carboxymethyl)-N-trans-(2-(bis(carboxymethyl)amino) cyclohexyl)-(4-nitrophenyl)alanine, with adenticity of 6, and N-(carboxymethyl)-N-(2-bis(carboxymehtyl)amino)phenyl)-(4-nitrophenyl)alanine, with a denticity of 6. ##STR11##

Further useful variants of the R₁ group include chelators in which R₁contains additional donor atoms capable of forming a coordinate bondwith the metal center. One such chelating agent according to the presentinvention isN-(1-carboxy-2-(p-nitrophenyl)-ethyl)-1,8-diamino-3,6-dioxaoctane-N,N',N'-triaceticacid. ##STR12##

In this structure, the two ether oxygen atoms can participate in metalbinding, together with the two amine nitrogen atoms and the fourcarboxylate oxygen atoms, leading to an overall denticity of eight.

The present invention further provides compounds wherein one or morecarboxymethyl groups in the polyaminopolycarboxylate chelator arereplaced by ortho-hydroxybenzyl groups. Such substituents comprise ametal binding moiety and present appropriate stereochemistry forchelation.N-((2-hydroxyphenyl)methyl)-N-(2-aminoethyl)-N'-((2'-hydroxyphenyl)methyl)-N'-(carboxymethyl)-(4-aminophenyl)alaninewith a denticity of 6 comprises an appropriate example of such acompound. Such a structure is believed to provide for improved stabilityof indium complexes relative to chelating agents of the prior art.##STR13##

The present invention also comprises macrocyclic chelating agentswherein R₂ and R₃ may be fused to form a hydrocarboy ring. Suchmacrocyclic chelating agents provide modified stability properties whichmay be desirable for certain applications. A compound exemplary of suchmacrocyclic chelating agents is N-((α-(3-(4-aminophenyl)propyl))carboxymethyl)-N',N'',N'''-(carboxymethyl)-1,4,8,11-tetraazacyclotetradecane shown below.##STR14##

Metal Ions

Metal ions which may be chelated according to the invention includegamma emitter isotopes which are useful for diagnostic scintigraphy.Indium¹¹¹ with a half-life of 2.8 days is particularly useful whileother suitable gamma emitters include gallium⁶⁷, gallium⁶⁸ andtechnetium^(99m). Materials according to the invention may be chelatedto beta radiation emitters which are useful as cytotoxic agents forradiotherapy. Such emitters include isotopes such as scadium⁴⁶,scandium⁴⁷, scandium⁴⁸, copper⁶⁷, gallium⁷², gallium⁷³, yttrium⁹⁰,ruthenium⁹⁷, palladium¹⁰⁰, rhodium^(101m), palladium¹⁰⁹, samarium¹⁵³,rhenium¹⁸⁶, rhenium¹⁸⁸, rhenium¹⁸⁹, gold¹⁹⁸, radium²¹² and lead²¹².

The chelating agents of the invention may also be used to bind alpharadiation emitting materials such as bismuth²¹², positron emitters suchas and zirconium⁸⁹, fluorescent members of the lanthanide series ofelements such as terbium and europium and of the transition series suchas ruthenium and paramagnetic materials such as gadolinium and iron. Inaddition the agents of the invention are also suitable for bindingnumerous other metal ions which may be useful for a variety of purposes,including those in which a catalytic property of the metal ion is ofutility. Iron, copper, vanadium, rhodium, platinum, palladium andtitanium are examples of metal ions useful in catalyzing a variety oforganic reactions, such as the cleavage of nucleic acids by theiron-catalyzed generation of hydroxyl free radicals.

Complexing of Metal Ions

Methods for forming chelating agent/metal ion conjugates are well knownto those of skill in the art. Complexes of the chelating agent and metalions may generally be formed by incubation of the chelatingagent/substrate conjugate with the metal ion in a buffered solution inwhich the conjugate is physiologically stable. Suitable buffers includethose with weak metal-binding properties, such as citrate, acetate orglycine. Appropriate concentrations, temperatures and pH may be selectedby one of skill in the art to ensure metal ions bind to the chelatingfunctionality rather than weak metal-binding sites on the substrates. Itis particularly desired that all solutions be maintained free of metalimpurities. After incubation for an appropriate period of time, unboundmetal ions may be separated, if necessary, from the substrate/chelatingagent/metal ion conjugate by a procedure such as gel filtration.

Substrate Reactive Functionalities

The substrate reactive moieties according to the present inventioncomprise those moieties capable of a specific binding reaction with atleast one functionality present in a substrate molecule which may be abiologically active substrate. If the substrate is a protein, suchmoieties may be reactive with side chain groups of amino acids making upthe polypeptide backbone. Such side chain groups include the carboxylgroups of aspartic acid and glutamic acid residues, the amino groups oflysine residues, the aromatic groups of tyrosine and histidine and thesulfhydryl groups of cysteine residues.

Carboxyl side groups presented by a substrate such as a polypeptidebackbone may be reacted with amine substrate reactive groups of thecompounds of the invention by means of a soluble carbodiimide reaction.Amino side groups presented by a substrate may be reacted with theisothiocyanate, isocyanate or halotriazine derivatives of this inventionto effect linkage of the chelator to the polypeptide. Alternatively,amino side groups on the substrate may be linked to compounds of thisinvention bearing amine substrate reactive groups by means ofbifunctional agents such as dialdehydes and imidoesters. Aromatic groupspresented by a substrate may be coupled to the chelating agents of thisinvention via the diazonium derivative. Sulfhydryl groups on substratemolecules may be reacted with maleimides or with haloalkyl substratereactive groups such as iodoacetamide. Free sulhydryl groups suitablefor such reactions may be generated from the disulfide bonds of proteinimmunoglobulin or may be introduced by chemical derivatization. Linkageto free sulfhydryl groups generated in the intra-heavy chain region ofimmunoglobulins does not interfere with the antigen binding site of theimmunoglobulin but may render the antibody incapable of activatingcomplement.

When the substrate is a glycosylated protein, an alternative to forminga linkage to the compounds of the present invention via the polypeptidebackbone is to form a covalent linkage with the carbohydrate side chainsof the glycoprotein according to the methods such as those of McKearn,et al., EPO 88,695. Thus, the carbohydrate side chains of antibodies maybe selectively oxidized to generate aldehydes which may then be reactedeither with amine substrate reactive groups to form a Schiff base orwith hydrazine, semicarbazide or thiosemicarbazide substrate reactivegroups, to give the corresponding hydrazone, semicarbazone orthiosemicarbazone linkages. These same methods may also be employed tolink the bifunctional chelators of this invention to non-proteinaceoussubstrates such as carbohydrates and polysaccharides.

An alternative substrate reactive moiety useful for linkage tocarbohydrates and polysaccharides without the necessity for prioroxidation is the dihydroxyboryl moiety, such as is present in the meta(dihydroxyboryl)phenylthiourea derivatives of the present invention.This moiety is reactive with substrates containing a 1,2-cis-diol,forming a 5-membered cyclic borate ester, and thus is of use with thosecarbohydrates, polysaccharides and glycoproteins which contain thisgroup. The dihydroxyboryl derivatives may also be used to link thechelators of this invention to ribonucleosides, ribonucleotides andribonucleic acids, since ribose contains a 1,2 -cis-diol group at the2',3' position, as disclosed by Rosenberg, et al., Biochemistry, 11,3623-28 (1972). Deoxyribonucleotides and DNA substrates may not belinked to the present chelators in this fashion as the 3' hydroxyl groupis absent. The latter substrates may, however, be conjugated toisothiocyanate derivatives of chelators by first forming an allylaminederivative of the deoxyribonucleotide as disclosed by Engelhardt, etal., EPO 97,373.

When the substrate to be linked with the chelators of this invention isan intact cell, either polypeptide reactive or carbohydrate reactivemoieties may be employed. Hwang and Wase, Biochim. Biophys. Acta, 512,54-71 (1978), disclose the use of the diazonium derivative of thebifunctional EDTA chelator of Sundberg, et al., J. Med. Chem., 17, 1304(1974), to label erythrocytes and platelets with indium-111. Thedihydroxyboryl moiety is reactive with a variety of bacteria, virusesand microorganisms, see Zittle, Advan. Enzym., 12 493 (1951) andBurnett, et al., Biochem. Biophys. Res. Comm., 96, 157-62 (1980).

According to the present invention, substrate reactive moieties includeamino (--NH₂), diazonium (--NN+), isothiocyanate (--NCS), isocyanate(--NCO), hydrazine (--NHNH₂), semicarbazide (--NHCONHNH₂),thiosemicarbazide (--NHCSNHNH₂), haloacetamide (--NHCOCH₂ X) includingchloro- , bromo- and iodoacetamide, azide (--N₃), aminoalkylurea(--NHCONH(CH₂)_(m) _(NH) ₂), aminoalkylthiourea (--NHCSNH(CH₂)_(m) NH₂),wherein m is from 1 to about 10, maleimide, halotriazine includingchloro-, bromo- and iodotriazine and meta-(dihyroxyboryl)phenylthiourea(--NHCSNHC₆ H₄ B(OH)₂). Other reactive moieties which may be suitablefor linking the chelating agents to substrates include disulfides,nitrenes, sulfonamides, carbodiimides, sulfonyl chlorides, benzimidates,--COCH₃ and --SO₃ H. The preferred substrate reactive moiety for anyparticular application of this invention will be dictated by the natureof the substrate and by its susceptibility to loss of biologicalactivity as a consequence of forming a given type of linkage. Bydefinition, the formation of any given linkage involves a chemicaltransformation of the substrate reactive moiety, X, into the conjugatedform of that moiety (hereafter the "residue" of X).

The reactive moieties of the invention are oriented at the meta orpreferably para position on a phenyl group which is attached by means ofan aliphatic spacer group to one of the carboxymethyl arms of thepolyaminopolycarboxylate chelating framework of the invention. Thespacer group may consist of from one to about ten carbon atoms, and maybe linear or branched alkyl or substituted alkyl provided such branchingor substituents do not interfere with the metal binding sites orsubstrate reactive groups. Linear alkyl linkers are neverthelesspreferred with Cl alkyl linkers particularly preferred.

Substrates Useful With the Present Invention

Substrate molecules which may be reacted with the chelating agents ofthe present invention include proteins, glycoproteins, peptides,poly(amino acids), lipids, carbohydrates, polysaccharides, nucleosides,nucleotides, nucleic acids, bile acids, drugs, inhibitors or intactcells. Suitable proteins include immunoglobulins, antigens, enzymes,components of the blood coagulation/anti coagulation system and variousbiochemically active molecules and receptors. Such proteins may bederived, for example, from genetically manipulated cells. According toone embodiment of the present invention, the bifunctional chelatingagents may be used to bind various types of antibodies including IgA,IgD, IgE, IgG and IgM. The antibodies may be directed against a varietyof antigenic determinants including those associated with tumors,histocompatibility and other cell surface antigens, bacteria, fungi,viruses, enzymes, toxins, drugs and other biologically active molecules.Antigens associated with tumors for which antibodies may be specificallyreactive include such antigens as are described in Zalcberg andMcKenzie, J. Clin. Oncology, Vol. 3; pp. 876-82 (1985) and include, butare not limited to, carcinoembryonic antigen (CEA), mucins such asTAG-72, human milk fat globule antigens and receptors such as the IL-2and transferrin receptors. Such antibodies may be monoclonal orpolyclonal or made by recombinant techniques such as described inMorrison, et al., Proc. Nat. Acad. Sci. U.S.A. 81, 6851-55 (1984).

Fragments of antibody molecules may also be bound including halfantibody molecules and Fab, Fab' or F(ab')₂ fragments. Nicolotti, EPO174,853 published Mar. 19, 1986 hereby incorporated by reference,discloses methods by which entire antibodies are treated to effect asite specific cleavage of the two heavy chains, removing the F_(c)portion at the carboxyl terminal ends of the heavy chains.

Substrates are reacted with the substrate reactive moieties of thechelating agents according to the methods disclosed above. Eachsubstrate may be bound by more than one chelating agent as may bedesired. The maximum extent of substitution on a substrate such as aprotein, however, is limited by the nature of glycosylation on theprotein or the number and location of reactive amino acid side chains onthe molecule. Where, as with antibodies, it is desired that theconjugated protein retain its biological activity, the extent ofsubstitution will be limited according to the nature and position oftarget glycosylation or amino acid residues both in the primary as wellas in the tertiary sequence of the protein and their degree ofinvolvement in the antigen binding site.

Other substrates contemplated by this invention include polysaccharidematrices which, when derivatized with chelators, provide means for theextraction of metals from metalloproteins and other metal containingsubstrates and for the affinity chromatography of proteins by themethods of Porath and Olin, Biochemistry, 22, 1621-30 (1983). Nucleicacids linked to the chelators of this invention may be used to monitorany nucleic acid hybridization reaction, as described by Engelhardt, etal., EPO 97,373. Bifunctional chelators linked to drugs may be used tofollow the uptake of that drug into tissues, as exemplified by use ofthe antibiotic drug bleomycin linked to the bifunctional EDTA derivativeof Sundberg, et al., J. Med. Chem., 17, 1304 (1974), as a means ofimaging tumors, see DeRiemer, et al., J. Med. Chem., 22, 1019-23 (1979);Goodwin, et al., J. Nucl. Med., 22, 787-92 (1981). In addition to drugs,other low molecular weight substances that target a particular organsystem may similarly be used to image that system. Thus cholic acid,which is known to target the hepatobiliary system in man, as describedby Boyd et al., J. Nucl. Med., 22, 720-725 (1981), may be used to imagethat system when conjugated to a bifunctional chelator of this inventionand labeled with either a gamma-emitting radiometal (forradioscintigraphic detection) or with a paramagnetic metal such agadolinium (for detection by means of Magnetic Resonance Imaging).Intact cells, such as erythrocytes and platelets, have been labelledwith radioisotopic metals by linkage to bifunctional chelators, asdescribed by Hwang and Wase, Biochim, Biophys. Acta, 512, 54-71 (1978),and such labelled cells may be used to detect areas of abnormalaccumulation in the body. Linkage of the compounds of this invention tolow molecular weight substances which themselves undergo a specificbinding reaction with a macromolecular biological molecule arecontemplated. Haner, et al., Arch. Biochem. Biophys., 231 477-86 (1984),disclose methods for linking EDTA to p-aminobenzamidine, a specificinhibitor of trypsin which binds strongly in the active site, providingan affinity label of use in probing that site. Schultz and Dervan, J.Amer. Chem. Soc., 105 7748-50 (1983), disclose the sequence specificdouble strand cleavage of DNA by iron complexes of conjugates formed bylinking EDTA to distamycin, an N methylpyrrole tripeptide which binds toDNA in a sequence specific manner.

The following examples illustrate methods for synthesis of variouschelating agents according to the invention. Examples 1 through 5describe the synthesis ofN-(carboxymethyl)-N-(2-(bis(carboxymethyl)amino)ethyl)-(4-aminophenyl)alanine (NH₂ -Compound A) and analogs presentingalternative substrate reactive groups. Examples 6 and 7 describesyntheses of N-(carboxymethyl)-N-(2-aminoethyl)-N'-(carboxymethyl)-N'-(2'-(bis-(carboxymethyl)amino)ethyl)-(4aminophenyl)alanine NH₂ -Compound B and its 4 isothiocyanatophenylanalogue. Example 8 describes synthesis of N(carboxymethyl)-N-(trans-(2-(bis(carboxymethyl)amino)cyclohexyl)-(4-nitrophenyl)alanine.

In examples 9 and 10, the 4 isothiocyanatophenyl chelating agent ofexample 3 was linked to an anti-CEA monoclonal antibody and theconjugate was compared with DTTA conjugates according to the prior art.In example 11, the 4-isothiocyanatophenyl chelating agent of example 7was conjugated to an anti CEA monoclonal antibody and evaluatedaccording to the methods of example 9. In example 12, the anti-CEAmonoclonal antibodies of example 9 were cleaved to provide F(ab')2fragments which were conjugated to the chelating agent of example 3. Inexample 13, the chelating agent of example 3 was conjugated tomonoclonal antibody B72.3 which is specifically reactive with a tumorassociated glycoprotein (TAG-72).

In example 14, the 4-(2-aminoethylthiourea)-phenyl chelating agent ofexample 5 was conjugated with polyglutamic acid. In examples 15, 16, 17and 18, biodistribution studies were conducted with the antibodyconjugates of examples 9, 11, 12 and 13, respectively.

Example 19 describes the synthesis of N-(1-carboxy-2-(p-nitrophenyl)-ethyl)-1,8-diamino-3,6-dioxaoctane-N,N'N'-triaceticacid, an 8-coordinate chelator presenting two ether oxyten donors inaddition to the nitrogen and carboxylate binding sites.

Example 20 describes the preparation of a conjugate formed between thebifunctional chelator of example 5 and cholic acid. In example 21, theconjugate of example 20 was labeled with indium-111 and biodistributionstudies were conducted in mice. Example 22 describes a gamma cameraimaging study in which the indium 111 labeled conjugate of example 21was used to image the hapatobiliary system of a rabbit.

EXAMPLE 1

In this example, N-(carboxymethyl)-N-(2-(bis(carboxymethyl)amino)ethyl)-(4-nitrophenyl)alanine (NO₂ -Compound A), apara-nitrophenyl substituted bifunctional chelating agent presenting twoamine and four carboxymethyl metal reacting groups according to theinvention, was synthesized by carboxymethylation of the monosubstituteddiamine obtained on reductive amination of 4-nitrophenylpyruvic acidwith ethylenediamine.

In order to produce the 4 nitrophenylpyruvic acid, the azalactone,5-keto2-methyl-4-(4'-nitroben-xylidine)-4,5-dihydro-oxazole was firstproduced by reaction at 100° C. for 2 hours of a solution comprising 7.0grams (60 mmol) of N-acetylglycine in 20 ml of acetic acid and 9.7 grams(64 mmol) of 4-nitrobenzaldehyde (Aldrich Chemical Co., Milwaukee,Wisc.) and 14.4 grams (176 mmol) sodium acetate in 21 ml of aceticanhydride. After cooling the mixture to 10° C., 100 ml of water wasadded with vigorous stirring and 12.9 grams of the azalactone wascollected by filtration.

In the next step, 7.9 grams (34 mmol) of5-keto-2-methyl-4-(4'-nitrobenzylidine)-4,5-dihydro-oxazole wasdissolved in 200 ml of acetic acid and was heated to 100° C. Five ml ofwater was then added and the mixture was stirred at 100° C. for anadditional 15 minutes. The solution was then allowed to cool slowly toroom temperature and 7.2 grams of α-acetamino-4-nitrocinnamic acid werethen isolated.

A suspension comprising 7.2 grams (29 mmol) ofα-acetamino-4-nitrocinnamic acid in 50 ml of 3 M HCl was stirred atreflux for 7 hours. The mixture was then cooled to 0° C. and 5.4 gramsof the resulting 4 nitro phenylpyruvic acid was collected by filtration,washed with cold H₂ O and dried under vacuum.

A solution comprising 15 grams (71.7 mmol) of 4-nitrophenylpyruvic acidin 500 ml methanol was added to a solution comprising 11.6 grams (87.2)ethylene diamine dihydrochloride in 100 ml water. The pH of theresulting mixture was then brought to 6.0 using 7 M sodium hydroxide. Tothe mixture was then added 7.86 grams (125 mmol) of sodiumcyanoborohydride and the pH was readjusted to 6.0 using 6 M HCl. Themixture was stirred at room temperature for 3 days and then 30 ml ofconcentrated HCl was added. After stirring for an additional 30 minutes,the solution was evaporated under vacuum until an orange residue wasrecovered: this was suspended in 250 ml of H₂ O and washed with ethylacetate (5×200 ml). The yellow aqueous layer was evaporated under vacuumleaving a yellow white residue which was identified as crudeN-(2-aminoethyl)-(4-nitrophenyl)alanine dihydrochloride. The crudeproduct was chromatographed on a Dowex 50X2-200 (H⁺ form) ion exchangecolumn, eluting initially with 700 ml of H₂ O and then with a 5% (v/v)solution of ammonium hydroxide. Ultraviolet light absorbingninhydrin-positive fractions were combined and evaporated under vacuumto provide a residue which was dissolved in ethanol. Addition ofconcentrated HCl to the solution caused precipitation of a dense whiteprecipitate which was collected by filtration, washed with ethanol anddiethyl ether and air dried to provide 6.56 grams of N(2-aminoethyl)-(4-nitrophenyl) alanine dihydrochloride.

Three grams (9.20 mmol) of the purifiedN-(2-aminoethyl)-(4-nitrophenyl)alanine dihydrochloride was then addedto a solution comprising 4.51 grams (32.5 mmol) of bromoacetic acid in25 ml of water and the mixture was heated to 45° C. The pH of thereaction mixture was brought to 10 by adding 7 M NaOH and the mixturewas stirred at 45° C. for 20 hours while the pH was maintained at 10 byperiodic addition of 7 M NaOH.

While intramolecular condensation leading to lactam formation did notpresent a problem during the reductive amination step to yield thediamine, the high temperatures used in the carboxymethylation reactionlead to formation of a lactam identified as a N,N'-bis(carboxymethyl)derivative. For this reason the cooled product was chromatographed toremove the lactam impurity on a Bio Rad AGI-X4 (formate form) ionexchange column (Bio Rad, Richmond, CA) by eluting successively with oneliter each of water, 3.5 M formic acid and 6.0 M formic acid. Thefractions were then evaluated by HPLC on a Waters Delta Prep 3000 systemusing a Waters μ-Bondpak C-18 column (0.39×30 cm) with 20% methanol and0.01 M triethylamine in acetic acid as a mobile phase. This analysisfound the lactam eluting with the 3.5 M formic acid and the desiredproduct eluting with the 6.0 M formic acid. Fractions containing theproduct were then combined and evaporated to dryness to yield 1.79 gramsofN-(carboxymethyl)-N-(2-(bis(carboxymethyl)amino)ethyl)-(4-nitrophenyl)alanine.

EXAMPLE 2

In this example, the 4-nitrophenyl substituted chelating agent of theinvention produced according to example 1 was converted to a4-aminophenyl substituted compound. A solution comprising 0.91 grams(2.13 mmol) of N-(carboxymethyl)-N-(2-(bis(carboxymethyl)amino)ethyl)-(4-nitrophenyl)alanine produced according to example 1, 100 ml ofwater and 15 ml of formic acid was hydrogenated in a Parr (Model 3911,Moline, Ill.) hydrogenator at room temperature and 35 psi for 2 hours inthe presence of 0.10 grams of 10% palladium on carbon as catalyst. Themixture was then filtered through celite to remove catalyst and thefiltrate was evaporated to dryness. The resulting residue was thendissolved in 50 ml of 4 M HCl and lyophilized to yield 0.87 grams of the4 aminophenyl substituted compound,N-(carboxymethyl)-N-(2-(bis(carboxymethyl)amino) ethyl)-(4-aminophenyl)-alanine trihydrochloride.

EXAMPLE 3

In this example, the 4-aminophenyl chelating agent of example 2 wasreacted to form the corresponding 4-isothiocyanatophenyl chelatingagent. Following the procedure of Meares, et al., Anal. Biochem., 142,68-78 . (1984), 0.68 grams (1.34 mmol) of the 4-aminophenyl product ofexample 2 was dissolved in 12 ml of 3 M HCl. To this was added 1.70 mlof 85% thiophosgene/CC14 (v/v)-(18.95 mmol), and the resultingsuspension stirred for 6 hours at room temperature. A 15 ml aliquot ofdiethylether was then added and the white precipitate formed wasfiltered off and dried under vacuum, to give 0.38 grams of the4-isothiocyanatophenyl substituted compound,N-(carboxymethyl)-N-(2-(bis(carboxymethyl)amino)ethyl)-(4-isothiocyanatophenyl) alanine dihydrochloride.

EXAMPLE 4

In this example, the 4-isothiocyanatophenyl chelating agent of example 3was reacted to form the corresponding 4-thiosemicarbazidophenylchelating agent. According to this procedure, 0.15 grams of the4-isothiocyanatophenyl product (0.29 mmol) was suspended in 15 ml ofwater and cooled in an ice bath. To this suspension was then added 0.255ml of triethylamine and 0.066 ml of 85% hydrazine hydrate (1.75 mmol)and the resulting mixture was stirred in the ice bath for 2 hours andthen at room temperature for a further 1 hour. The mixture wasevaporated to dryness under vacuum and the residue dissolved in 20 ml of4 M HCl. This solution was again evaporated to dryness under vacuum andthe resulting residue was chromatographed on a Bio Rad AGl-X4 ionexchange column (chloride form), eluting successively with 100 ml eachof water and 4 M HCl. The 4 M HCl eluate was evaporated to dryness undervacuum to yield 0.15 grams ofN-(carboxymethyl)-N-(2-(bis(carboxymethyl)amino)ethyl)-(4-thiosemicarbazidophenyl)alaninetrihydrochloride in the form of a white solid.

EXAMPLE 5

In this example, the 4-isothiocyanatophenyl chelating agent of example 3was converted to the corresponding 4-(2-aminoethylthiourea)phenylderivative, providing a reactive aliphatic amine group linked to thephenylthiourea substituent by a 2-carbon spacer.

First, a mono-protected ethylenediamine derivative was prepared byadding a solution of 6.0 grams of di-t-butyldicarbonate (27.5 mmol) in300 ml of THF drop wise over 3 hours to a stirred solution of 40.5 gramsof ethylenediamine (674 mmol) in 150 ml of methanol which was cooled inan ice bath. The resulting mixture was stirred in the ice bath for afurther 1 hour, then was warmed to room temperature. The residueobtained on evaporation of this solution to dryness was thenchromatographed on a silica column (Kieselgel, 70-230 mesh, E. Merck,Darmstadt, W. Germany) eluted with (20:0.5:79.5) methanol:ammoniumhydroxide:methylene chloride. Fractions containing the desired productwere identified by thin layer chromatography on silica plates (WhatmanCo., Clifton, N.J.) developed in the same solvent mixture used to elutethe column (R_(f) =0.6). These fractions were combined and evaporated todryness under vacuum to yield 4.1 grams (93%) ofN-(t-butoxycarbonyl)ethylenediamine in the form of a yellow oil.

In the next step, a solution of 0.39 grams ofN-(t-butoxycarbonyl)ethylenediamine (2.4 mmol) in 5 ml of DMF was addedto a mixture of 0.34 grams of the 4-isothiocyanatophenyl product ofexample 3 (0.66 mmol) and 0.35 ml of triethylamine (2.5 mmol) in 7 ml ofDMF cooled to 0° C.. The resulting mixture was stirred at 0° C. for afurther 15 minutes and then at room temperature for 48 hours. At thatpoint, 2 ml of water were added and the mixture stirred for a further 6hours then evaporated to dryness under vacuum. The residue was dissolvedin 20 ml of water and this solution was washed with three 25 ml aliquotsof methylene chloride. The aqueous layer was then lyophilized and theresulting tan colored solid chromatographed on a Bio Rad AGl-X4 column(formate form) eluted successively with 150 ml of water, 200 ml of 3.5 Mformic acid and 200 ml of 7 M formic acid. The 7 M formic acid eluatewas evaporated to dryness to yield 0.23 grams (58%) ofN-(carboxymethyl)-N-(2-(bis(carboxymethyl)amino) ethyl)-(4-(N'-(2'-(N''-t-butoxycarbonyl))aminoethylthiourea) phenyl)alanine. 0.17grams of this material (0.28 mmol) were then de-protected by stirring in5.0 ml of trifluoroacetic acid at room temperature for 6 hours,protected from atmospheric moisture. The solvent was then evaporated,the residue dissolved in 10 ml of 2 M HCl and this solution wasevaporated to dryness under vacuum. The resulting residue waschromatographed on a Bio-Rad AGl-X4 column (formate form) elutedsuccessively with 100 ml of water then 50 ml each of 1 M, 2 M, 3 M and 4M formic acid. Ultraviolet absorbing fractions from the formic acideluates were combined and evaporated to dryness under vacuum. Theresulting residue was dissolved in 100 ml of 4 M HCl and evaporated todryness under vacuum to yield 0.14 grams (81%) ofN-(carboxymethyl)-N-(2-(bis(carboxymethyl)amino)ethyl)-(4-(N'-(2-aminoethyl) thiourea)phenyl) alaninetrihydrochloride.

EXAMPLE 6

In this example, N (carboxymethyl)N-(2-aminoethyl)-N'-(carboxymethyl)-N'-(2'-(bis(carboxymethyl)amino)ethyl)-(4-aminophenyl)alanine(NH2-Compound B), a para-aminophenyl substituted bifunctional chelatingagent presenting three amine and five carboxymethyl metal reactinggroups according to the invention, was synthesized by reduction andsubsequent acid hydrolysis of the material obtained oncarboxymethylation of the product formed by reductive amination of 4nitrophenylpyruvic acid with N⁴-(N,N-diethylcarboxamidomethyl)diethyl-enetriamine.

In order to produce N⁴ -(N,N-diethylcarboxamidomethyl)diethylenetriamine, the doubly protected triamine, N¹,N⁷-bis(t-butoxycarbonyl)diethylenetriamine, was first prepared by addingover 30 minutes a solution of 26.0 grams of2-(t-butoxycarbonyloxyimino)-2-phenyl-acetonitrile (105.6 mmol, AldrichChemical Co., Milwaukee, Wisc.) in 500 ml of THF to a stirred solutioncontaining 5.44 grams of diethylenetriamine (52.7 mmol, Aldrich) and15.97 grams of triethylamine (157.8 mmol) in 100 ml of THF cooled in anice bath. After stirring for 2 more hours in the ice bath and then for afurther 1 hour at room temperature, the THF was removed by evaporationunder vacuum to give a green oil. This was dissolved in 200 ml of ethylacetate and the resulting solution washed with 1000 ml of cold aqueousNaOH solution 5% (w/v). The organic layer was dried over anhydroussodium sulfate then evaporated to dryness under vacuum. The resultingresidue was purified by preparative HPLC using a Waters Delta Prep 3000system (Millipore Corp., Milford, Mass.) equipped with a Prep-Pak 500silica column and eluted with methanol:(2.5:97.5) methylene chloride.Fractions containing the desired product were identified by thin layerchromatography on silica plates (Whatman) developed in the same solventsystem. These fractions were combined and evaporated to dryness undervacuum to yield 11.3 grams (71%) of N¹,N⁷ -bis(t-butoxycarbonyl)diethylenetriamine as a white solid.

In the next step, a diethylacetamide group was substituted at thecentral, unprotected nitrogen of the diethylenetriamine framework byrefluxing together for 48 hours a mixture of 10.40 grams of N¹,N⁷-bis(t-butoxycarbonyl)diethylenetriamine (34.29 mmol), 5.13 grams of2-chloro-N,N-diethylacetamide (34.29 mmol, Aldrich) and 3.54 grams oftriethylamine (34.98 mmol) in 200 ml of ethanol. After cooling to roomtemperature, the solvent was evaporated under vacuum and 200 ml of ethylacetate added to the residue. The resulting mixture was filtered and thefiltrate was washed with 1000 ml of 5% aqueous sodium carbonatesolution. The organic layer was dried over anhydrous sodium sulfate thenevaporated to dryness under vacuum, yielding a yellow oil. This waspurified by preparative HPLC, using the Prep-Pak 500 silica column andeluting with (5:95) methanol:methylene chloride, fractions containingthe desired product again being identified by thin layer chromatographyon silica plates developed in the same solvent mixture used to elute thecolumn. These were combined and evaporated to dryness to give 10.70grams (75%) of N¹,N⁷ -bis(t-butoxycarbonyl)-N⁴-(N,N-diethylcarboxamidomethyl)diethylene triamine as a yellow oil. A10.0 gram aliquot of this material (24.0 mmol) was de protected bystirring in 75 ml of trifluoroacetic acid for 2 hours at roomtemperature. The reaction mixture was then evaporated to dryness, theresidue dissolved in 100 ml of 4 M HCl and again evaporated to drynessunder vacuum to yield 7.45 grams (95%) ofN4-(N,N-diethylcarboxamidomethyl)-diethylenetriamine trihydrochloride asa white solid.

The subsequent reductive amination step was achieved by adding asolution comprising 5.0 grams of the purified N⁴-N,N-diethylcarboxamidomethyl)-diethylenetriamine trihydrochloride(15.35 mmol) in 15 ml of water to a solution comprising 3.14 grams of4-nitrophenylpyruvic acid (15.01 mmol, prepared as described inexample 1) in 50 ml of methanol. The pH of the resulting mixture wasthen brought to 6.0 using 7 M sodium hydroxide and 1.45 grams of sodiumcyanoborohydride (23.07 mmol) was added. The pH of the mixture wasreadjusted to 6.0 using 6 M HCl then stirring was continued for afurther 3 days at room temperature before 15 ml of concentrated HCl wasadded. Evaporation to dryness under vacuum gave an orange residue, whichwas suspended in 200 ml of water and washed with 1200 ml of ethylacetate. Evaporation of the aqueous layer to dryness gave a yellow-whiteresidue which was purified by preparative HPLC on the Prep-Pak 500silica column eluting with (20:80 ) methanol:0.01 M triethylammoniumacetate. Fractions containing the desired product were identified bythin layer chromatography on silica plates developed in (20:80) ammoniumhydroxide:95% ethanol. These fractions were combined and the buffersalts were removed by re applying this material to the preparative HPLCcolumn, and eluting with (25:75) methanol:water. Fractions containingthe desired product were again identified by thin layer chromatography,combined and evaporated to dryness. The resulting residue was dissolvedin 50 ml of 4 M HCl and evaporated to dryness under vacuum to yield 1.96grams ofN-(2-aminoethyl)-N'-(N'',N''-diethylcarboxamidomethyl)-N'-(2'-aminoethyl)-(4-nitrophenyl)alanine trihydrochloride as a white solid (25%).

A 1.0 gram aliquot of the purifiedN-(2-amino-ethyl)-N'-N'-(N'',N''-diethylcarboxamidomethyl)-N'-(2-amino-aminoethyl)-(-4-nitrophenyl)alaninetrihydrochloride (1.93 mmol) was then added to a solution comprising1.16 grams of bromoacetic acid (8.33 mmol) in 20 ml of water and themixture was heated to 45° C. The pH of the reaction mixture was broughtto 12 by adding 7 M NaOH and the mixture was stirred at 45° C. for 24hours while the pH was maintained at 12 by periodic addition of 7 MNaOH. The mixture was then cooled to room temperature and the pHadjusted to 1 using concentrated HCl. The mixture was washed with 150 mlof ethyl acetate then the pH of the aqueous layer was brought to 12 byadding 7 M NaOH. The resulting solution was applied to a Bio-Rad AGl-X4column (formate form) which was eluted first with 500 ml of water thenwith 750 ml of 1.2 M formic acid. ractions containing the desiredproduct were identified by thin layer chromatography on silica platesdeveloped in (20:80) ammonium hydroxide:95% ethanol. These fractionswere combined and evaporated to dryness to give a yellow oil, which wasredissolved in 50 ml of 4 M HCl. Evaporation to dryness of this solutionyielded 0.60 grams (45% ofN-(carboxymethyl)-N(2-aminoethyl)-N'-(N',N''-diethylcarboxamidomethyl)-N'-(2'-(bis(carboxymethyl)amino)ethyl)(4 nitrophenyl)alanine trihydrochloride in the form of a white solid.

In the next step, a solution comprising 1.1 grams ofN-(carboxymethyl)-N-(2-aminoethyl)-N'-(N'',N''-diethylcarboxamidomethyl)-N'-(2'-(bis(carboxymethyl)amino)ethyl)-(4-nitrophenyl)alanine trihydrochloride (1.59 mmol), 300 mlof water and 30 ml of formic acid was hydrogenated at room temperatureand 35 psi for 2 hours in the presence of 0.11 grams of 10% palladium oncarbon as catalyst. The mixture was then filtered through celite toremove the catalyst and the filtrate was evaporated to dryness. Theresulting residue was then dissolved in 100 ml of 4 M HCl and evaporatedto dryness under vacuum to yield 1.1 grams (98%) ofN-(carboxymethyl)-N-(2-aminoethyl)-N'-(N'',N''-diethyl-carboxamidomethyl)-N'-(2'-(bis(carboxymethyl)amino) ethyl)-(4-aminophenyl)alanine tetrahydrochloride.

A solution comprising 1.0 grams ofN-(carboxymethyl)-N-(2-aminoethyl)-N'-(N'',N''-diethylcarboxamidomethyl)-N'-(2'-(bis(carboxymethyl)amino)ethyl)(4-amino-phenyl)alanine tetrahydrochloride (1.43 mmol) in 100 ml of 6 MHCl was refluxed for 60 hours. After cooling the reaction mixture toroom temperature, the solvent was removed by evaporation under vacuumand the resulting residue was chromatographed on a Bio-Rad AGl-X4 column(formate form) eluted successively with 400 ml each of water, 0.1 Mformic acid, 0.2 M formic acid and 0.3 M formic acid. The desiredproduct eluted in 0.3 M formic acid, the relevant fractions beingidentified by thin layer chromatography on silica plates developed in(20:80) ammonium hydroxide:95% ethanol. These fractions were combinedand evaporated to dryness. The resulting residue was dissolved in 50 mlof 4 M HCl and evaporated to dryness under vacuum to yield 0.2 grams(21%) ofN-(carboxymethyl)-N-(2-aminoethyl)-N'-(carboxymethyl)-N'-(2'-(bis(carboxymethyl)amino)ethyl)-(4-aminophenyl)alaninetetrahydrochloride in the form of a white solid.

EXAMPLE 7

In this example, the 4 aminophenyl chelating agent of example 6 wasconverted to the corresponding 4-isothiocyanatophenyl chelating agent. Asolution comprising 0.10 grams ofN-(carboxymethyl)-N-(2-amino-ethyl)-N'-(carboxymethyl)-N'-(2'-(bis(carboxymethyl)amino)ethyl)-(4-aminophenyl)alaninetetrahydrochloride (0.16 mmol), 4 ml of 3 M HCl and 0.2 ml of 85%thiophosgene/carbon tetrachloride (v/v) (2.23 mmol) was stirred at roomtemperature for 6 hours. An additional 5 ml of carbon tetrachloride wasthen added and the aqueous and organic phases were separated. Theaqueous layer was evaporated to dryness under vacuum to yield 0.093grams (92%) ofN-(carboxymethyl)-N-(2-aminoethyl)-4-isothiocyanatophenyl) alaninetrihydrochloride.

EXAMPLE 8

In this example,N-(carboxymethyl)-N-(trans-(2-(bis(carboxymethyl)amino)cyclohexyl))-(4-nitrophenyl)-alanine,a para nitrophenyl substituted bifunctional chelating agent presentingtwo amine and four carboxymethyl metal reacting groups with the aminenitrogen atoms linked through a hydrocarbyl ring according to theinvention, was synthesized by reduction and subsequentcarboxymethylation of the imine obtained by the Schiff base condensationof 4-nitrophenylpyruvic acid with trans-1,2-diaminocyclohexane.

In the first step, 2.96 grams of trans-1,2-diaminocyclohexane (25.9mmol, Aldrich) were added at room temperature to a solution comprising5.0 grams of 4-nitrophenylpyruvic acid (23.9 mmol, prepared as inexample 1) dissolved in 100 ml of methanol. The mixture was stirred atroom temperature for 22 hours, then the resulting precipitate wasfiltered off, washed with methanol and dried under vacuum to yield 6.78grams of 2 (trans-(2-aminocyclohexyl)imido)-3-(4-nitrophenyl) propionicacid.

In the next step, a solution comprising 0.24 grams of sodium borohydride(6.29 mmol) in 4 ml of ethanol was added to a suspension comprising 1.83grams of 2-(trans-2 -aminocyclohexyl)imido)-3-(4-nitrophenyl)-propionicacid (6.0 mmol) and 0.2 grams of sodium hydroxide in 35 ml of ethanol inan ice bath. The reaction mixture was stirred in the ice bath for 30minutes then at room temperature for a further 30 minutes. The pH of thereaction mixture was then brought to 6 by the addition of formic acid,causing a precipitate to form. This was filtered off and washed withethanol. The filtrate plus washings were then evaporated to drynessunder vacuum and the resulting residue was dissolved in water andbrought to pH 10. This solution was washed with 50% ethyl acetate:50%diethyl ether, then the aqueous phase was chromatographed on a Bio-RadAGl-X4 column (formate form) eluted first with 200 ml of water andsubsequently with 500 ml of 0.05 M formic acid. Ultraviolet lightabsorbing and ninhydrin-positive fractions were combined and evaporatedto dryness under vacuum. Trituration of the resulting residue withmethanol gave a white solid which on drying under vacuum provided 0.91grams of N-(trans-(2-aminocyclo-hexyl))-(4-nitrophenyl)alanine.

A 0.3 gram aliquot of the purifiedN-(trans-(2-aminocyclohexyl))-(4-nitrophenyl)alanine (0.99 mmol)dissolved in 20 ml of water were then added to a solution comprising0.87 grams of bromoacetic acid (6.29 mmol) in 4 ml of water. The pH ofthe reaction mixture was adjusted to 13 by the addition of 0.2 grams ofsodium hydroxide then 0.04 grams of sodium iodide were added and theresulting mixture was heated to 57° C and stirred for 22 hours. Aftercooling to room temperature, the pH of the reaction mixture was restoredto 13 by the addition of 5% aqueous sodium hydroxide. This solution wasthen chromatographed on a Bio Rad AGl-X4 column (formate form) elutedsuccessively with 75 ml of water, 100 ml of 0.2 M formic acid, 500 ml of1.0 M formic acid and 250 ml of 5.0 M formic acid. The desired producteluted in the 5 M formic acid, ultra violet light absorbing fractionsbeing combined and evaporated to dryness to yield 0.02 grams ofN-(carboxy-methyl)-N-(trans-(2-(bis(carboxymethyl)amino)cyclohexyl))-(4-nitrophenyl)alanine.

EXAMPLE 9

In this example, the 4-isothiocyanatophenyl chelating agent(NCS-Compound A) produced according to example 3 was linked to ananti-CEA monoclonal antibody and the resulting conjugate compared withthose obtained by a prior art procedure whereby a diethylenetriaminetetraacetate (DTTA) residue is introduced into a protein molecule byreaction of that protein with the bicyclic anhydride of DTPA, asdisclosed by Hnatowich, et al., J. Immunol. Meth., 65, 147 (1983). Theantibody was an immunoglobulin of the IgG₁ sub-type which binds to CEAwith an affinity constant in excess of 10⁹ L.mol⁻¹ and does not reactwith non specific cross reacting antigen 1 (NCA-1). The antibody wasproduced in BALB/c mice and was isolated from ascites by affinitychromatography on protein A sepharose CL-4B (Sigma Chemical Co., St.Louis, Mo.). After exhaustive dialysis against 0.1 M KH₂ PO₄ /0.15 MNaCl/0.05 M HEPES, pH 7.2, the antibody concentration was adjusted to1.0 mg/ml and the solution stored at 2°-8° C. until needed. Antibodyconcentrations were determined by the Bradford dye binding assay(Bio-Rad Laboratories, Richmond, Calif.), which was performed accordingto the manufacturer's directions. All buffers were prepared using waterfrom a MILLI-Q system (Millipore Cowporation, Bedford, Mass.) purifiedto a resistivity of 18 megohm or greater. Antibody solutions wereconcentrated where necessary by ultrafiltration, using anultrafiltration cell equipped with a membrane having a nominal molecularweight cut off of 10,000 daltons (Amicon Corporation, Danvers, Mass.).

The antibody was conjugated to the 4-isothiocyanatophenyl chelatingagent of example 3 according to the general procedure described byMeares, et al., Anal. Biochem., 142, 68 (1984) the disclosure of whichis hereby incorporated by reference. Specifically, 10 milligrams of themonoclonal antibody were buffer exchanged by dialysis overnight at fromabout 2° to about 8° C. against 0.1 M KH₂ PO₄ /0.1 M NaHCO3 (pH 8.5).After adjusting the antibody solution to a concentration of 1.0 mg/ml,N-(carboxymethyl)-N-(2-(bis(carboxymethyl)amino)ethyl)-(4-isothiocyanatophenyl) alanine dihydrochloride was addedsuch as to give a 500-fold molar excess of chelator relative toantibody. The chelator/antibody solution was then incubated for 3 hoursat 37° C. and the resulting conjugate (hereafter "anti-CEA NCS CompoundA") dialysed for 48 hours at from about 2° to about 8° C. against 0.05 Msodium citrate (pH 6.0). The conjugate solution was then stored at -20°C. until needed.

The average chelator substitution in terms of moles of chelator per moleof antibody in the antibody chelator conjugate was determined by theradiocobalt binding assay of Meares, et al., Anal. Biochem., 142, 68(1984). The results of this procedure showed that theanti-CEA-NCS-Compound A conjugate contained an average 2 of 4 chelatorsper antibody.

The formation of antibody chelator inter molecular aggregates wasmeasured by SDS-polyacrylamide gel electrophoresis on 10% slab gelsaccording to the methods of Laemmli, et al., Nature, 227 680 (1970).Gels were stained with Coomassie Blue and examined for the presence ofbands other than those characteristic of light and heavy immunoglobulinchains. The presence of cross-linked material was indicated by thepresence of a band which barely migrated into the gel and was wellseparated from the two bands arising from monomeric antibody chelatorconjugates, as described by Paik, et al., J. Nucl. Med., 24 1158 (1983).This procedure revealed no cross linking in the anti-CEA-NCS-Compound Aconjugate.

In order to compare the bifunctional Compound A conjugate of the presentinvention with chelate immunoconjugates of the same anti-CEA monoclonalantibody prepared by the prior art method, two DTTA conjugates wereprepared: the first having a comparable level of substitution in termsof moles of chelator per mole of antibody, the second having acomparable low level of cross-linking. According to this procedure, theantibody was buffer-exchanged by dialysis overnight against a solutioncomprising 0.1 M NaHCO₃ /0.05 M HEPES (pH 8.2). The antibodyconcentration was then adjusted to 1.0 mg/ml and the resulting solutionwas cooled to 0°-5° C. and stirred while a saturated solution of thebicyclic anhydride of DTPA (Sigma Chemical Co., St. Louis, Mo.) in DMSOwas added drop wise to the antibody solution. The pH of the reactionmixture was closely monitored throughout the addition process and wasmaintained at 8.2 by addition of 0.05 M NaOH. After mixing was complete,the solution was stirred at 0°-5° C. for an additional 15 minutes alongwith continued addition of base to maintain the pH at 8.2. The resultingconjugate was then dialysed for 2 days at from about 2 to about 8° C.against multiple changes of 0.05 M sodium citrate (pH 6.0) and thenstored at -20° C. until needed. When this reaction was run with a 600:1molar excess of bicyclic DTPA anhydride to antibody, the resultingconjugate contained an average of 5 chelators per antibody molecule(hereafter "anti-CEA-DTTA(5.0)"), and thus was comparable in its levelof substitution to the anti-CEA-NCS-Compound A conjugate. However, thegel electrophoresis procedure detected virtually no monomericimmunoglobulin in the anti-CEA DTTA(5.0) conjugate. When the couplingreaction was run using a 100:1 molar excess of bicyclic DTPA anhydrideto antibody, a conjugate was obtained which was shown by electrophoresisto contain only small amounts of cross linked material, the averagesubstitution by radiocobalt binding assay being found to be 0.5chelators per antibody (hereafter "anti-CEA DTTA(0.5)").

EXAMPLE 10

In this example, the conjugates according to example 9 were evaluatedusing an enzyme linked immunosorbent assay (ELISA) procedure todetermine the degree of immunoreactivity retained by the conjugated formcompared with the free form. Ninety-six well microtiter plates(Immulon™, Dynatech Laboratories, Inc., Arlington, Va.) were coated withpurified CEA by incubating in each well 0.1 ml of a solution comprising1.0 ug of the antigen in 10 mmol Tris (pH 7.4). After incubationovernight at room temperature, the wells were emptied and washed twicewith deionized water. The wells were then overcoated by incubating for 2hours at room temperature with a solution of bovine serum albumin (SigmaChemical Co., St. Louis, Mo.), 0.1% w/v in 0.1 M phosphate bufferednormal saline (pH 7.4). The overcoated plates were stored until use at2°-8° C. with a 0.1 ml aliquot of the overcoating solution in each well.Immediately prior to use, the plate was emptied and the wells werewashed five times with deionized water.

Tandem ELISA assays were then performed on each antibody chelatorconjugate and on the native (underivatized) antibody from which theconjugate had been prepared. Both assays were run on the same microtiterplate, employing successive 2-fold dilutions of solutions adjusted to aninitial protein concentration of 2.5 q/ml. Duplicate wells were run ateach concentration for each antibody preparation.

According to the assay procedure, a solution of bovine serum albumin(1.0% w/v) and Tween (Sigma, 0.1%, by volume) in 0.05 ml of 0.1 Mphosphate buffered normal saline (pH 7.4) was added to each wellfollowed by 0.05 ml of the antibody preparation. The plate was thencovered and incubated at 37° C. for 1 hour and then emptied and washedfive times with deionized water. A 0.1 ml aliquot of solution comprising0.06 ug/ml goat anti-mouse antibody conjugated to horseradish peroxidase(Kirkegaard & Perry Labs, Inc., Gaithersburg, Md.) 1.0% bovine serumalbumin, 0.1% Tween and 0.1 M phosphate buffered normal saline (pH 7.4)was then added to each well. The plate was then covered and incubated at37° C. for 1 hour. After being emptied and washed five times withdeionized water, a 0.1 ml aliquot of orthophenylenediamine solutionprepared according to the manufacturer's directions from apre-formulated kit (Abbott Laboratories, North Chicago, Ill.) was addedto each well. The plate was then incubated in the dark at roomtemperature for 15 minutes and 0.1 ml of 2 M H₂ SO₄ was then added toeach well to quench the enzymatic reaction. The color generated in eachwell was read at 490 nm using a microtiter plate reader (Minireader II,Dynatech).

Antibody titration curves were then prepared by plotting the meanoptical density of duplicates at 490 nm against antibody concentrationand the curve for the conjugate compared to that for the underivatizedantibody. A semi-quantitative estimate of the immunoreactivity retainedafter labelling could be obtained by expressing the absorbance of theconjugate as a percentage of absorbance of the native antibody at 50%titration.

The results of the ELISA assays shown in FIGS. 1a, 1b and 1c show thatthe anti-CEA DTTA(0.5) conjugate retains good immunoreactivity comparedwith the unconjugated antibody. On the other hand, the anti-CEADTTA(5.0) conjugate retains little activity as compared with theunconjugated antibody. The ELISA plot indicated that theanti-CEA-NCS-Compound A conjugate with an average of 4.0 chelators perantibody molecule maintained a significant level of immunoreactivity,close to that of the underivatized antibody. As the degree ofsubstitution is increased, anti-CEA-NCS-Compound A conjugates fail todisplay any significant loss of immunoreactivity until a level ofapproximately 10 chelators per antibody is reached. Beyond this point,there appears a progressive loss of ability to bind CEA.

EXAMPLE 11

In this example, the 4-isothiocyanatophenyl chelating agent(NCS-Compound B) of example 7 was conjugated to the same anti-CEAmonoclonal antibody employed in example 9. This was achieved by dialysisof 10 milligrams of the antibody against 0.1 M KH₂ PO₄ /0.1 M NaHCO₃ (pH8.5) overnight at from about 2° to about 8° C. The antibodyconcentration was adjusted to 10 mg/ml then solidN-(carboxymethyl)-N-(2-aminoethyl)-N'-(carboxymethyl)-N'-(2'-(bis(carboxymethyl)amino)ethyl)-(4-isothiocyanatophenyl)alaninetrihydrochloride was added such as to provide a 25-fold molar excess ofchelating agent relative to the immunoglobulin. Gentle mixing caused thesolid chelating agent to dissolve and produced a clear homogeneoussolution which was incubated at 37° C. for 3 hours. The resultingconjugate solution was dialysed for 24 hours at from about 2° to about8° C. against 0.05 M sodium citrate/0.01 M DTPA (pH 6.0) then for afurther 24 hours at from about 2 to about 8° C. against 0.05 M sodiumcitrate (pH 6.0). Analysis of this anti-CEA monoclonal monoclonalantibody conjugate (hereafter "anti-CEA-NCS-Compound B") by the methodsdescribed in examples 9 and 10 revealed no inter molecular aggregation,an average substitution of 13 chelators per antibody and completeretention of immunoreactivity.

EXAMPLE 12

In this example, the anti-CEA monoclonal antibody of examples 9, 10 and11 was cleaved to provide the corresponding F(ab')₂ fragment, which wasthen conjugated to the 4-isothiocyanatophenyl (NCS-Compound A) chelatingagent of example 3. Digestion of the antibody was carried out accordingto the general procedure described in Parham et al., J. Immunology, 131,2895 (1983). Specifically, 10 milligrams of the antibody at aconcentration of 2 mg/ml in phosphate buffered saline, pH 7.2, wereadded to 1.66 ml of 1.0 M citric acid, pH 3.5. To this solution was thenadded 0.83 ml of a solution of pepsin (0.25 mg/ml) in phosphatebuffered:saline:citric acid (9:1 v/v), pH 3.7, followed by a further1.66 ml of phosphate buffered saline, pH 7.2. The resulting mixture wasincubated at 37° C. for 16 hours then the pepsin was inactivated by theaddition of 1.66 ml of 1.0 M Tris. The solution was passed over aprotein A-sepharose column (Pharmacia, N.J.) to remove intactimmunoglobulin and F_(c) fragments, then the eluate was chromatographedon a Bio-Gel P-100 column (Bio-Rad) to separate the immunoglobulinfragments from the pepsin. The desired F(ab')₂ fragments eluted in thevoid volume from this column.

In the next step, the F(ab')₂ fragments derived from the anti-CEAmonoclonal antibody were dialysed overnight at from about 2° to about 8°C. against 0.1 M KH₂ PO₄ /0.1 M NaHCO₃ (pH 8.5). The concentration ofthe fragments was adjusted to 1.33 mg/ml, then solid 30N-(carboxymethyl)N-(2-(bis(carboxymethyl)amino)ethyl)-(4-isothiocyanatophenyl)alaninedihydrochloride was added to a total of 2.0 mg of fragments such as toprovide a final 250 fold molar excess of chelating agent to antibodyfragment. After the solid chelating agent had dissolved, the resultingsolution was incubated for 3 hours at 37° C. then dialysed for 24 hoursat from about 2° to about 8° C. against 0.05 M sodium citrate/0.01 MEDTA (pH 6.0) then for a further 24 hours at the same temperatureagainst 0.05 M sodium citrate, pH 6.0. Analysis of the resultingconjugate (hereafter "anti- CEA-F(ab').sub. 2 -NCS-Compound A") by themethods described in example 9 showed an average substitution of 1chelator per F(ab')₂ fragment and no aggregated material byelectrophoresis. The immunoreactivity of the fragment and its chelatedconjugate were assessed by a minor modification of the proceduredescribed in example 10, wherein the goat anti-mouse antibody conjugatedto horseradish peroxidase used for color development in example 10 wasreplaced with a horseradish peroxidase conjugate of a goat antibodyspecific for the light chains of mouse immunoglobulin (CooperBiomedical, Malvern, Pa.). When assayed in this manner, theimmuno-reactivity of the chelate conjugate was identical to that of theunderivatized fragment.

EXAMPLE 13

In this example, the monoclonal antibody B72.3 which recognizes atumor-associated glycoprotein (TAG-72) and has been extensivelydescribed by Schlom et al., Int. J. Cancer, 29, 539 (1982), wasconjugated to the 4-isothiocyanatophenyl chelating agent of example 3.The antibody was first dialysed overnight at from about 2° to about 8°C. against 0.1 M KH₂ PO₄ /0.1 M NaHCO₃ (pH 8.5) at an antibodyconcentration of 10 mg/ml. A 10 mg aliquot of the resulting antibodysolution was then mixed with 1 milligram ofN-(carboxymethyl)-N-(2-(bis(carboxymethyl)amino)ethyl)-(4-isothiocyanatophenyl)alanine trihydrochloride dissolvedin 62 microliters of the same phosphate/bicarbonate buffer. Theresulting molar ratio of chelator:antibody was 20:1. After incubation at37° C. for 3 hours, the conjugate was dialysed for 24 hours at fromabout 2° to about 8° C. against 0.05 M sodium citrate/0.01 M EDTA (pH6.0) then for a further 24 hours against 0.05 M sodium citrate, pH 6.0.Analysis of the resulting immunoconjugate by the methods described inexample 9 revealed an average substitution of 1 chelator per antibodyand a complete absence of cross-linked material. The immunoreactivity ofB72.3 conjugates was determined by a minor modification of the ELISAprocedure described in example 10, wherein the micro-titer plate wascoated not with CEA but with bovine submaxillary mucin (a readilyavailable antigen which cross reacts with B72.3, Cooper Biomedical), thecoating solution containing 10 micrograms of mucin in 10 mmol TRIS, pH7.4. Otherwise, the procedure given in example 10 was followed exactly,except that washing steps employed 0.1 M KH₂ PO₄ /0.15 M NaCl, pH 7.4,in place of deionized water. When assayed in this fashion, theimmunoreactivity of the chelate conjugate B72.3 (hereafter"B72.3-NCS-Compound A") was found to be approximately 50% that of theunderivatized antibody.

EXAMPLE 14

In this example, the 4-(2-aminoethylthiourea)-phenyl chelating agent ofexample 5 (aminoethyl NCS-Compound A) was conjugated to the nonproteinaceous substrate poly(glutamic acid). 2.3 milligrams of thesodium salt of poly(glutamic acid) (0.16 mmol, Sigma Chemical Co., St.Louis, Mo.) containing an average of 90 glutamate residues per moleculeand having an average molecular weight of 14,000 daltons was dissolvedin 0.3 ml of dry dimethylformamide. 1.5 milligrams of 4-methylmorpholine(14.8 mmol) was added and the solution was cooled in an ice bath. Tothis stirred solution was added 2.0 milligrams of isobutylchloroformate(14.8 mmol) and the mixture was stirred in the ice bath for 1 hour. Afurther 4.5 milligrams of 4-methylmorpholine (44.4 mmol) were thenadded, followed by 10.0 milligrams ofN-(carboxymethyl)-N-(2-(bis(carboxymethyl)amino)ethyl)-(4-(N'(2-aminoethyl)thiourea)phenyl)alanine trihydrochloride(14.8 mmol) and the resulting mixture was stirred overnight at roomtemperature. After diluting the reaction mixture to a total volume of5.0 ml using 0.1 M NaH₂ PO₄, pH 7.0, it was dialysed exhaustivelyagainst the same buffer, using dialysis tubing having a nominalmolecular weight cut off of 2,000 daltons (Spectrum Medical Industries,Inc., Los Angeles, Calif.), then against deionized water. The resultingsolution was lyophilized to yield a conjugate containing between 20 and30 chelate residues per poly(glutamicacid) chain.

EXAMPLE 15

In this example, anti-CEA immunoconjugates were labelled with gammaemitting indium¹¹¹ and their biodistributions were studied in nude micebearing xenografts of human colorectal carcinoma line LS174T expressinghigh levels of CEA. The antibody-chelator conjugates included theanti-CEA-DTTA(0.5) conjugate, the anti-CEA DTTA(5.0) conjugate and theanti-CEA-NCS-Compound A conjugate according to example 9. Each waslabelled with indium¹¹¹ according to the procedure wherein a 0.1 mlaliquot of the antibody-chelator conjugate at a concentration of 1.0mg/ml in 0.05 M sodium citrate (pH 6.0) was transferred to a plasticmicro test tube and the pH was adjusted to 4.5-5.0 by addition of 6 MHCl. Six μl of carrier-free ¹¹¹ InCl₃ in 0.04 M HCl 3(ca. 50-400 mCi/ml,typically about 80 mCi/ml, NEN-DuPont) was added to the conjugatesolution and the pH was readjusted to 7.0 by addition of 6 M NaOH. Afterincubating the solution for 30 minutes at room temperature, unboundindium^(I11) was separated from antibody bound radioactivity by thecentrifuged gel filtration column method described by Meares, et al.,Anal. Biochem., 142, 68 (1984). The radiolabelled conjugate, whicheluted from a Sephadex G-50 microcolumn on centrifugation for 2 minutesat 100 g, was diluted into normal saline to a final concentration of 10ug/ml. ELISA assays performed as described in example 10 showed no lossof immunoreactivity during the indium¹¹¹ labelling procedure.

Prior to injection into animals a 20 ul aliquot of this solution wasincubated for 20 minutes at room temperature with 10 ul of a 0.1 M EDTAsolution. The specificity of labelling was then assessed by thin layerchromatography according to the method of Meares, et al., which wascarried out under the same conditions as in the radiocobalt bindingassay. In all cases, greater than 90% of the indium¹¹¹ activity remainedbound to the antibody in the face of the EDTA challenge. Specificactivities of 2-3 mCi ¹¹¹ In/mg of protein were typically achieved.

Female athymic nude mice (nu/nu, BALB/C background, Charles RiverBiotechnology Services, Inc., Wilmington, Mass.) were prepared forbiodistribution studies by subcutaneous injection in the right rearflank with a suspension of LS174T human colorectal carcinoma cells(1.25×10⁶ -2.5×10⁶ cells in 0.1 ml normal saline). Within 1-2 weeks,solid tumors developed and reached a size of 0.4-0.8 grams.

Animals were then randomized into treatment groups, typically 5 mice pergroup, and injected intraveneously via the tail vein with 1.0 ug of oneof the three radiolabelled antibody-conjugates in 0.1 ml normal saline.At various times after injection, animals were sacrificed by cervicaldislocation and all their internal organs were removed, weighed andcounted in a gamma counter (AUTO-LOGIC®gamma counter, AbbottLaboratories). Weighed aliquots of blood, muscle and skin were alsocounted, as was the residual carcass. The tail was counted separately tocheck for extravasation at the injection site and, when radioiodinatedantibodies were used in control studies, the head was counted separatelyto evaluate thyroid uptake. A 0.1 ml aliquot of the injectate wascounted at the same time as the tissues and the radioactivity measuredin each tissue was then expressed as a percentage of this injected doseper gram of tissue.

Control studies were conducted involving implantation of a CEAantigen-negative tumor on the left rear flank of animals bearing anantigen positive tumor on their right flank. The MIA ATCC No. CRL 1420human pancreatic carcinoma line was used for this purpose because itdoes not express appreciable amounts of CEA. The uptake of the anti-CEAantibody into the LS174T xenografts was some 8 to 10 fold greater thanthat of the MIA carcinoma.

Other control studies were conducted with anti-alpha-fetoprotein IgG,monoclonal antibodies labelled with iodine¹²⁵. The labellings wereconducted using the chloramine-T method to specific activities of 5 to10 mCi/mg. These studies showed that uptake of non-CEA specificantibodies was an order of magnitude less than that of the anti-CEAantibodies. The injected doses of protein and the dissection andcounting procedures in these studies were identical to those describedabove for the indium labelled reagents. Because time-course studies ofanti-CEA antibody uptake into the LS174 tumor showed that tumor/bloodratios reached a maximum 2 days after intravenous injection of antibody,that was the interval chosen for studies with the chelator conjugates.

The results of the biodistribution studies are presented in tables 1 and2. In table 1, the anti-CEA-NCS-Compound A conjugate of example 3 iscompared with the prior art anti-CEA DTTA(5.0) conjugate. The lowerimmunoreactivity and extensive cross linking present in theanti-CEA-DTTA(5.0) conjugate is correlated with a lower tumor uptake andelevated liver accumulation. The mean tumor/liver ratio in animals giventhe anti-CEA-5NCS-Compound A conjugate according to the invention was2.50±1.24. This was significantly higher (p<0.05 according to atwo-tailed Student's t test) than the anti-CEA-DTTA(5.0) conjugate whichgave a mean tumor/liver ratio of 0.92±0.28.

                  TABLE 1                                                         ______________________________________                                                  % of Injected Dose                                                            per Gram of Tissue                                                              anti-CEA-NCS-                                                                             anti-CEA-                                             Organ       Compound A  DTTA (5.0)                                            ______________________________________                                        Tumor       21.6 (3.2)  13.6 (3.8)                                            Blood       10.3 (1.6)  7.8 (2.5)                                             Liver       10.3 (5.3)  15.6 (4.5)                                            Spleen      4.1 (0.8)   5.6 (2.1)                                             Lungs       5.5 (0.9)   5.8 (2.2)                                             Heart       4.7 (1.5)   3.5 (0.5)                                             Gut         1.5 (0.5)   1.8 (0.3)                                             Kidney      5.9 (0.8)   7.0 (1.3)                                             Muscle      1.5 (0.2)   1.2 (0.4)                                             Skin        3.8 (0.5)   3.7 (0.7)                                             ______________________________________                                         All values are shown as mean (± SD) for n = 5.                        

                  TABLE 2                                                         ______________________________________                                                  % of Injected Dose                                                            per Gram of Tissue                                                              anti-CEA-NCS-                                                                             anti-CEA-                                             Organ       Compound A  DTTA (5.0)                                            ______________________________________                                        Tumor       21.6 (3.2)  28.3 (4.9)                                            Blood       10.3 (1.6)  10.4 (2.7)                                            Liver       10.3 (5.3)  11.1 (1.2)                                            Spleen      4.1 (0.8)   7.1 (1.2)                                             Lungs       5.5 (0.9)   6.2 (1.2)                                             Heart       4.7 (1.5)   4.2 (0.7)                                             Gut         1.5 (0.5)   2.2 (0.7)                                             Kidney      5.9 (0.8)   11.7 (1.2)                                            Muscle      1.5 (0.2)   1.7 (0.4)                                             Skin        3.8 (0.5)   6.1 (0.7)                                             ______________________________________                                         All values are shown as mean (± SD) for n = 5.                        

In table 2, the anti-CEA-NCS-Compound A conjugate of example 9 wascompared with the anti-CEA-DTTA(0.5) conjugate. Even though there was an8-fold difference in the level of substitution, the conjugates werecompared at equivalent levels of immunoreactivity. There was nosignificant difference in mean tumor/liver uptake ratios (2.56±0.43 forthe anti-CEA-DTTA(0.5) conjugate versus 2.50±1.24 for the conjugate ofthe invention although absolute tumor uptake was significantly higherfor the anti-CEA-DTTA (0.5) conjugate. At the same time, however, uptakeinto most other body organs was higher for the anti-CEA-DTTA(0.5)conjugate as reflected in the whole body retention of radioactivity at48 hours which was significantly higher (p<0.0111) for theanti-CEA-DTTA(0.5) conjugate than for the conjugate of the invention. Asa consequence, the absolute amount of radioactivity in the tumorexpressed as a percentage of whole body radioactivity, which is animportant index for radioimmunotherapy modeling, was not significantlygreater for the anti-CEA-DTTA(0.5) conjugate than for theanti-CEA-NCS-Compound A conjugate of the invention.

                  TABLE 3                                                         ______________________________________                                        (Indium.sup.111 -anti-CEA Conjugates)                                                               Whole Body  Tumor                                                             Activity    Activity                                                          (% of       (% Whole                                    Indium.sup.111                                                                           Tumor Wt.  Injected    Body                                        Conjugate  (Grams)    Dose)       Activity)                                   ______________________________________                                        DTTA (0.5) 0.53 (0.28)                                                                              76 (6)      21.5 (3.4)                                  DTTA (5.0) 0.61 (0.43)                                                                              66 (5)      11.2 (5.0)                                  NCS-Cmpd A 0.40 (0.21)                                                                              54 (5)      19.1 (4.7)                                  ______________________________________                                    

Table 3 shows the tumor uptake of indium¹¹¹ labelled anti-CEA conjugatesin relation to whole body retention of activity in nude mice bearingLS174T tumors. Tumor uptake and whole body activity were measured atsacrifice 48 hours after intravenous injection of the conjugates intothe nude mice. Tumor weights of the animals at sacrifice averagedapproximately equal and were within a fairly narrow range. This issignificant because large LS174T tumors tend to become necrotic withconsequent low uptake while subcutaneous LS174T xenografts less thanabout 100 mg in weight are frequently poorly vascularized also withconsequent low uptake rates. Consequently, tumor size can effect tumoruptake values even when these have been normalized to a unit weightbasis.

EXAMPLE 16

In this example, the biodistribution of the anti-CEA-NCS-Compound Bconjugate of example 11 was studied using an indium¹¹¹ radiolabel andthe nude mouse xenograft model described in example 15. The anti-NCS-Compound B conjugate of example 11 was labelled with indium¹¹¹ by theprocedure described in example 15 and injected into mice bearing LS174Txenografts at a dose of 1.0 micrograms of conjugate per mouse.Biodistribution data, obtained at 48 hours post injection as describedin example 15, is shown in table 4.

                  TABLE 4                                                         ______________________________________                                                    % of Injected Dose                                                Organ       per Gram of Tissue                                                ______________________________________                                        Tumor       19.7 (5.3)                                                        Blood       6.1 (1.9)                                                         Liver       3.9 (0.8)                                                         Spleen      4.3 (0.7)                                                         Lungs       3.7 (0.7)                                                         Heart       4.1 (1.4)                                                         Gut         1.0 (0.3)                                                         Kidney      3.3 (0.7)                                                         Muscle      2.0 (0.4)                                                         Skin        2.5 (0.8)                                                         ______________________________________                                         All values are shown as mean (± SD) for n = 5.                        

When the biodistribution data for the anti-CEA-NCS-Compound B conjugateare compared with those for the conjugates of example 15, it is evidentthat while the tumor uptake of anti-CEA-NCS-Compound B is no greaterthan that seen with anti-CEA-NCS-Compound A and anti-CEA-DTTA(0.5), boththe liver uptake and blood levels of radioactivity at 48 hours arestrikingly lower with anti-CEA-NCS-Compound B. As a result, thetumor/liver uptake ratio for animals treated with anti-CEA-NCS-CompoundB (5.3±1.7) is significantly higher than that for animals given eitherthe anti-CEA-NCS-Compound A conjugate of this invention (2.5±1.2,p<0.01) or the prior art anti-CEA-DTTA(0.5) conjugate (2.6±0.4, p0.001). Similarily, the tumor/blood uptake ratio foranti-CEA-NCS-Compound B (3.40±1.15) is significantly higher than thatfor anti-CEA-NCS-Compound A (2.15±0.49, p<0.05).

                  TABLE 5                                                         ______________________________________                                                    Whole Body                                                                    Activity                                                                      (% of      Tumor Activity                                         Tumor Wt.   Injected   (% Whole Body                                          (Grams)     Dose)      Activity)                                              ______________________________________                                        0.93 (0.41) 59 (6)     29.8 (13.6)                                            ______________________________________                                         All values are mean (± SD) for n = 5.                                 

Table 5 shows the tumor uptake of indium¹¹¹ labelledanti-CEA-NCS-Compound B in relation to whole body retention of activityin nude mice bearing LS174T tumors at 48 hours post injection. Althoughcomparison of the data in table 5 with those in table 3 of example 15suggests that the absolute amount of radioactivity in the tumorexpressed as a percentage of whole body radioactivity is higher for theanti-CEA-NCS-Compound B conjugate than for the conjugates of example 15,this difference is not statistically significant and probably reflectsthe greater mean tumor weight in the given anti-CEA-NCS-Compound B.

EXAMPLE 17

In this example, the biodistribution of the anti-CEA-F(ab')₂-NCS-Compound A of example 12 was xenograft using an indium¹¹¹radiolabel and the nude mouse xenograft model described in example 15.The conjugate was labelled with indium¹¹¹ by the procedure described inexample 15 and injected into mice bearing LS174T xenografts at a dose of1.0 microgram per mouse. Tables 6 and 7 present biodistribution dataobtained 48 hours post injection as described in example 15.

                  TABLE 6                                                         ______________________________________                                                    % of Injected Dose                                                Organ       per Gram of Tissue                                                ______________________________________                                        Tumor       4.71 (1.01)                                                       Blood       0.33 (0.08)                                                       Liver       1.01 (0.06)                                                       Spleen      1.03 (0.21)                                                       Lungs       0.98 (0.04)                                                       Heart       1.01 (0.14)                                                       Gut         0.41 (0.10)                                                       Kidney      5.63 (1.41)                                                       Muscle      0.80 (0.15)                                                       Skin        0.67 (0.14)                                                       ______________________________________                                         All values are mean (± SD) for n = 5.                                 

                  TABLE 7                                                         ______________________________________                                                     Whole Body                                                                    Activity                                                                      (% of      Tumor Activity                                        Tumor Wt.    Injected   (% Whole Body                                         (Grams)      Dose)      Activity)                                             ______________________________________                                        0.36 (0.26)  14.1 (1.7) 10.7 (4.9)                                            ______________________________________                                         All values are means (± SD) for n = 5.                                

Other than the high kidney uptake, reflecting accelerated renalclearance, the most prominent difference between the biodistribution ofthe antibody fragment and that of the corresponding conjugate of theintact antibody (example 15) is, as anticipated, the much lower level ofradioactivity remaining in the blood at 48 hours. This results in atumor/blood ratio (14.5±3.3) which greatly exceeds those seen with anyof the intact antibody conjugates of examples 15 and 16. This ability toachieve a high level of tumor contrast relative to the blood backgroundand thus to image a tumor at early time periods post injection is amajor advantage offered by antibody fragments. A potential disadvantagewhen attempting therapy with cytotoxic radiometals is that the absoluteuptake of radioactivity into the tumor is substantially lower than thatseen with conjugates of the intact antibody. This remains true even whenthe tumor activity is expressed as a percentage of the whole bodyradioactivity at 48 hours, despite the latter being some 4-fold lowerthan the whole body retention of activity in animals given conjugates ofthe intact antibody.

EXAMPLE 18

In this example, the B72.3-NCS-Compound A conjugate of example 13 waslabelled with indium¹¹¹ and a time course study was conducted in nudemice bearing a TAG-72 positive tumor on one flank (LS174T, see Keenan etal., J. Nucl. Med., 25, 1197 (1984)) and a TAG 72 negative xenograft onthe opposing flank (the melanoma line, A375, was used for this purpose).This model has been described in detail by Colcher et al., Cancer Res.,44, 5744 (1984) and by Brechbiel et al., Inorg. Chem., 25, 2772 (1986).The method used to label the B72.3-NCS-Compound A conjugate withindium111 and the biodistribution procedures were as described inexample 15. Each animal received 1.0 microgram of the conjugate via tailvein injection at time zero, then groups of mice were sacrificedserially at 24, 72, 120 and 168 hours post-injection. The data appear intable 8.

                  TABLE 8                                                         ______________________________________                                        Tissue  24 hrs.   72 hrs.    120 hrs.                                                                              168 hrs.                                 ______________________________________                                        LS174T  16.3 (5.4)                                                                              16.0 (3.4) 13.2 (2.2)                                                                            12.1 (2.7)                               A375    3.8 (2.5) 9.9 (3.5)  9.0 (3.1)                                                                             9.0 (2.1)                                Blood   16.0 (8.2)                                                                              6.1 (0.8)  4.2 (0.8)                                                                             3.1 (0.9)                                Liver   4.2 (0.8) 4.5 (0.6)  5.7 (1.3)                                                                             4.4 (1.0)                                Spleen  3.2 (0.8) 4.0 (0.4)  4.6 (1.2)                                                                             4.7 (2.1)                                Kidney  3.3 (0.8) 4.4 (1.1)  5.3 (1.1)                                                                             5.5 (1.4)                                ______________________________________                                         All values are mean ± SD for n = 5.                                   

Tumor uptake was maximal at 24 hours and slowly declined thereafter.Blood levels of radioactivity declined more precipitously, with theresult that tumor/blood ratios increased progressively throughout theperiod of the study. Liver, spleen and kidney uptakes were modest at 24hours and did not increase significantly at the later time points.Uptake into all other tissues was unremarkable.

EXAMPLE 19

In this example, a para-nitrophenyl substituted bifunctional derivativeof the chelating agent ethylene glycol bis(2-aminoethyl ether)N,N,N',N'-tetraacetic acid (EGTA) was prepared by the reductivealkylation of 1,8-diamino-3,6-dioxaoctane with p-nitrophenylpyruvic acidfollowed by carboxymethylation of the resulting product.

To a solution containing 0.21 grams of 4-nitro-phenylpyruvic acid (1.0mmol) in 5 ml of methanol was added a solution of 0.15 grams of1,8-diamino-3,6-dioxaoctane (1.01 mmol) in 1 ml of water. The resultingdeep red solution was treated with 4M hydrochloric acid until the pHreached 6.0. Then 0.1 grams of sodium cyanoborohydride was added. Theresulting reaction mixture was stirred at room temperature at pH 6.0 for4 days, then was acidified to a pH of 1.0 by the addition ofconcentrated hydrochloric acid. The resulting mixture was evaporated todryness under vacuum and the residue dissolved in 100 ml of water. Thisaqueous solution was extracted with three 100 ml aliquots of ethylacetate. Next, the aqueous phase was concentrated under vacuum, frozenin a isopropanol/dry ice bath and lyophilized. The lyophilized solid wasdissolved in 5 ml of water and the pH of this solution was adjusted to3.0 by addition of 4M hydrochloric acid. The solution was then appliedto a Dowex 50X2-200 cation exchange column (bed size 25 grams) which waseluted successively with water, 1M hydrochloric acid, 2M hydrochloricacid and 4M hydrochloric acid. The desired product eluted in the 4Mhydrochloric acid fractions. These fractions were combined, concentratedunder vacuum and subjected to four cycles of dilution with 50 ml ofwater and reconcentration under vacuum, to remove excess hydrochloricacid. The residue after complete removal of solvents under vacuum wasredissolved in water, frozen in an isopropanol/dry ice bath andlyophilized to afford 0.075 grams of the desired product,N-(1-amino-3,6-dioxaoctyl) 4 nitrophenylalanine.

0.06 grams of the above intermediate,N-(1-amino-3,6-dioxaoctyl)-4-nitrophenylalanine (0.145 mmol), wasdissolved in a solution of 0.44 ml of 1M sodium hydroxide and 0.05 ml ofDMF. This solution was then added dropwise to a stirred solution of 0.06grams of bromoacetic acid (0.43 mmol) in 0.44 ml of 1M sodium hydroxidesolution. Then an additional 0.44 ml of 1M sodium hydroxide was added tothe solution. The resulting reaction mixture was stirred at about 80° C.for 2.5 hours, and then was cooled to room temperature and acidified topH 1 by the addition of concentrated hydrochloric acid. The solution wasconcentrated under vacuum and the residue was subjected to four cyclesof redissolution in 100 ml of water and re evaporation under vacuum, toremove excess hydrochloric acid. The resulting oily residue wasdissolved in 10 ml of water, frozen in an isopropanol/dry ice bath andlyophilized. This gave 0.19 grams of a light beige colored powder whichwas dissolved in a minimum volume of water and the pH adjusted to 8using sodium hydroxide. The resulting solution was applied to a Bio RadAGl-X4 anion exchange column (formate form, bed size 5 grams) which wassuccessively eluted with water, 1M formic acid, 2M formic acid, 3Mformic acid and 5M formic acid. The product eluted in the 1M formic acidfractions. The fractions were combined, concentrated to dryness undervacuum and lyophilized to yield 0.04 grams of the desired product,N-(1-carboxy-2-(p-nitrophenyl)-ethyl)-1,8-diamino-3,6-dioxaoctane-N,N',N'-triaceticacid.

EXAMPLE 20

In this example, a conjugate was formed between cholic acid, which is abile acid containing a side chain bearing an aliphatic carboxylic acidgroup and the 4-(2-aminoethylthiourea)phenyl bifunctional derivative ofethylenediaminetetraacetic acid described in Example 5. The conjugatewas obtained by first forming an active ester derivative of cholic acidand then reacting this with the aliphatic amine substituent in thebifunctional chelator.

The active ester was prepared by dissolving 1.1 grams of cholic acid(2.4 mmol) and 0.35 grams of N-hydroxysuccinimide (3.04 mmol) in amixture of 20 ml of THF and 5 ml of acetonitrile and cooling theresulting solution in an ice bath. A solution of 0.47 grams of1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (2.4 mmol)in 5 ml of THF was then added dropwise, followed by 0.34 grams oftriethylamine (2.4 mmol). The reaction mixture was stirred at roomtemperature for 17 hours. The solvents were then removed by evaporationunder vacuum. The resulting residue was dissolved in 150 ml ofchloroform and then extracted successively with four 100 ml aliquots ofcold water, four 100 ml aliquots of cold 1M hydrochloric acid, three 100ml aliquots of cold saturated sodium bicarbonate solution. The organicphase was then separated and dried over anhydrous sodium sulfate. Thedessicant was filtered off and the filtrate evaporated to dryness undervacuum to yield 1.18 grams of the desired compound in the form of awhite solid.

A solution of 31 milligrams of N-(carboxymethyl) N(2-(bis(carboxymethyl)amino)ethyl)-(4-(N'(2-aminoethyl)thiourea)phenyl)alanine trihydrochloride (0.05 mmol, prepared asdescribed in Example 5) and 36 milligrams of triethylamine (0.36 mmol)in 1 ml of THF was stirred at room temperature as a solution of 25.5milligrams of the cholic acid active ester (0.05 mmol) in 0.25 ml of DMFwas added dropwise. This reaction was stirred at room temperature forsix days in a tightly stoppered flask. The solvents were then removed byevaporation under vacuum and the residue was dissolved in 5 ml of water.The pH of this solution was adjusted to about 8 using 1M sodiumhydroxide, then it was applied to a Bio-Rad AGl-X4 anion exchange colume(formate form, bed size 7 grams). The column was eluted successivelywith water, 1M formic acid, 3M formic acid, 4M formic acid, 4.5M formicacid, 5M formic acid and 1M hydrochloric acid. The desired materialeluted in the 5M formic acid fractions. These fractions were combinedand evaporated to dryness under vacuum. The resulting residue wassubjected to four cycles of redissolution in 100 ml of 4M hydrochloricacid and re-evaporation to dryness. The residue from this process wasdissolved in 150 ml of water and lyophilized to give a sticky yellowsolid. This was redissolved in 50 ml of water, frozen in anisopropanol/dry ice bath and again lyophilized to yield the desiredcholic acid-EDTA conjugate in the form of a light beige powder, yielding30 milligrams.

EXAMPLE 21

In this example, the cholic acid EDTA conjugate prepared in Example 20was labelled with indium-111 and its biodistribution was determined inmice.

20 microliters of 1.0M sodium acetate buffer, pH 6, were placed in amicro test tube together with 20 microliters of indium-111 chloridesolution (13.4 micro-Curies per microliter, Atomic Energy of Canada,Ltd.) and 40 microliters of a stock solution of the cholic acid EDTAconjugate (prepared as in Example 20) obtained by dissolving 2milligrams of the conjugate in 2 ml of 0.1M sodium acetate buffer, pH 6.The contents of the tube were mixed and allowed to stand at roomtemperature for 30 minutes. Forty microliters of the reaction mixturewere then applied to a small column (bed volume 300 microliters)containing Chelex resin (Sigma Chemical Co., 50-100 mesh) and unboundindium was removed by centrifugation of the column, as described byMeares et al., Anal. Biochem., 142, 68 (1984). The column eluent was0.1M sodium acetate, pH 6 (600 microliters). The indium-111 labeledcholic acid-EDTA conjugate was collected in the column eluate aftercentrifugation and found to have a specific activity of 8.6 microcuriesper microliter.

Five BALB/c mice were anesthetized by an i.p. injection of nembutal andthen were given i.v. injections via the tail vein of 100 microliters ofthe indium-111-EDTA-cholic acid conjugate. The mice were sacrificed bycervical dislocation at 2 hours post injection and the biodistributionof indium 111 activity was determined. These data appear in Table 9.

                  TABLE 9                                                         ______________________________________                                                      % of injected dose                                              Tissue        per gram of tissue*                                             ______________________________________                                        Blood         0.99                                                            Liver         1.01                                                            Heart         0.39                                                            Kidney        2.9                                                             G.I. Tract    6.2                                                             Gall Bladder  56.0                                                            Spleen        0.40                                                            Lungs         1.2                                                             Muscle        0.26                                                            ______________________________________                                         *Values shown are means for n = 5.                                       

These data indicate that a significant fraction of the injected dose wascleared through the liver with subsequent biliary excretion into thegut. Substantial activity was present in the gut at 2 hours with markedaccumulation in the gall bladder. The residual activity seen in thekidneys probably indicates that fraction of the injected activity whichwas not cleared by the liver was rapidly excreted into the urine.

EXAMPLE 22

In this example, the ability of the cholic acid-EDTA-indium-111conjugate to function as an imaging agent for the hepatobiliary systemwas evaluated in a rabbit model.

The cholic acid EDTA conjugate prepared in Example 20 was labeled withindium 111 as described in Example 21. The resulting preparation had aspecific activity of 1.69 mCi/ml. A 1.0 mCi dose of this material (0.59ml) was injected into the left marginal ear vein of a female New Zealandrabbit which had been fasted for two days prior to the study. Fiveminutes after receiving the injection, the rabbit was anesthetized withInnovar Vet and a series of planar gamma camera images were obtained at5 minute intervals over the following hour. Additional images wereacquired at 80, 95, 110, 125 and 140 minutes post-injection. The rabbitwas then allowed to regain conciousness. At 25 hours post injection, therabbit was again anesthetized and a further gamma camera image wasobtained.

The resulting images showed rapid uptake of the radiolabel into theliver, such that the first image obtained at 10 minutes post injectionshowed intense liver localization. Subsequently, the activity wasrapidly cleared from the liver with focal accumulations of labelbecoming apparent in the lower abdomen at 20-25 minutes post-injectionand no observable activity remaining in the liver at 1 hour. Activityalso was apparent in the kidneys at the 10 minute time point, but thiscleared rapidly such that there was no discernable kidney activity at 35minutes post injection. The urinary bladder showed intense activity atall early time points, but not in the 25 hour image at which time theanimal had emptied its bladder. Activity in the 25 hour image wasdiffusely distributed throughout the G.I. tract.

The results of this study establish the utility of the cholicacid-EDTA-In-111 conjugate as a radiopharmaceutical for imaging of thehepatobiliary system. A substantial portion of this conjugate is rapidlyextracted from circulation by the liver, from which it is then excretedthrough the bile duct into the gut, allowing the potentialidentification of physiological defects in these structures by NuclearMedicine procedures.

From the above examples, it is obvious to those of skill in the art thatthe conjugates and methods of the invention are useful for imaging oflocalized concentrations of antigens by external photoscanning such asdescribed in Goldenberg, et al. In such a method, the conjugate isintroduced into a patient and the body of the patient is scanned forconcentrations of the conjugate. It should also be apparent that theconjugates of the invention may be utilized in in vitro diagnosticmethods such as immunoassays or nucleic acid hybridization assays. Indiagnostic methods such as sandwich hybridization techniques, conjugatesaccording to the invention comprising indicator means are useful inindicating the presence of analytes. Conjugates and methods of theinvention are also useful in therapeutic methods wherein anantibody-metal ion conjugate in which the metal ion emits cytotoxicradiation is introduced into a patient such that cytotoxic radiation maybe directed to tumors while minimizing the toxic effects to healthytissues. Consequently, only such limitations should be placed on theinvention as appear in the following claims.

We claim:
 1. A compound characterized by having the structure ##STR15##wherein X is meta or para and is nitro or is selected from the groupconsisting of

    ______________________________________                                        NH.sub.2,           (AMINO)                                                   NN+,                (DIAZONIUM)                                               NCS,                (ISOTHIOCYANATE)                                          NCO,                (ISOCYANATE)                                              NHNH.sub.2,         (HYDRAZINE)                                               NHCSNHNH.sub.2,     (THIOSEMI-                                                                    CARBAZIDE)                                                NHCOCH.sub.2 Cl,    (CHLORO-                                                                      ACETAMIDE)                                                NHCOCH.sub.2 Br,    (BROMOACETAMIDE)                                          NHCOCH.sub.2 I,     (IODOACETAMIDE)                                           N.sub.3,            (AZIDE)                                                   NHCONH(CH.sub.2).sub.m NH.sub.2,                                                                  (AMINOALKYLUREA)                                          NHCSNH(CH.sub.2).sub.m NH.sub.2,                                                                  (AMINOALKYL-                                                                  THIOUREA)                                                 NHCONHNH.sub.2,     (SEMICARBAZIDE)                                            ##STR16##          (MALEIMIDE)                                                ##STR17##          (HALOTRIAZINE)                                            and                                                                            ##STR18##          (META-(DIHYDROXY- BORYL)PHENYL- THIOUREA)                 ______________________________________                                    

wherein Y is selected from the group consisting of Cl, Br, and F;wherein Z is selected from the group consisting of Cl, Br, F, OH, andOCH₃ ; wherein m=1 to 10; wherein n=0 to 10; wherein R₁ is selected fromthe group consisting of: ##STR19## wherein q=2 or 3r=2 or 3, and s=2 or3; wherein R₂, R₃, R₄, R₅ and R₆ are the same or different and areselected from the group consisting of:hydrogen CH₂ CO₂ H ortho-CH₂ CH₆H₄ OH, and whereint=2 or 3, u=2 or 3, and v=2 or 3 wherein R₇ and R₈ areselected from the group consisting of:hydrogen, --CH₂ CO₂ H, andortho-CH₂ C₆ H₄ OH.
 2. The compound according to claim 1 wherein X isselected from the group consisting of:

    --NO.sub.2,

    --NH.sub.2,

    --NCS,

    --NHCSNHNH.sub.2,

    --NHCOCH.sub.2 Br and

    --NHCSNH(CH.sub.2).sub.2 NH.sub.2.


3. The compound according to claim 1 wherein X is para substituted. 4.The compound according to claim 1, wherein R₂, R₃, R₄, R₅ and R₆ are thesame or different and are selected from the group consisting of hydrogenand CH₂ CO₂ H and wherein R₂ and R₃ are not fused to form a hydrocarbylring.
 5. The compound according to claim 1 wherein R₁ is selected fromthe group consisting of:

    --(CH.sub.2).sub.2 --,

    --[(CH.sub.2).sub.2 O(CH.sub.2).sub.2 O(CH.sub.2).sub.2 ]--,

    --[(CH.sub.2).sub.2 N(CH.sub.2 CO.sub.2 H)(CH.sub.2).sub.2 ]--,

    [(CH.sub.2).sub.2 N(CH.sub.2 CO.sub.2 H)(CH.sub.2).sub.2 N(CH.sub.2 CO.sub.2 H)(CH.sub.2).sub.2 ]--, ##STR20##


6. The compound according to claim 1 wherein n=1.
 7. The compoundaccording to claim 1 wherein X is para and is selected from the groupconsisting of:

    --NO.sub.2,

    --NH.sub.2,

    --NCS,

    --NHCSNHNH.sub.2 and

    --NHCSNH(CH.sub.2).sub.2 NH.sub.2

wherein n=1, wherein R₁ is selected from the group consisting of

    --(CH.sub.2).sub.2 --,

    --[(CH.sub.2).sub.2 O(CH.sub.2).sub.2 ]--,

    --[(CH.sub.2).sub.2 N(CH.sub.2 CO.sub.2)(CH.sub.2).sub.2 ]--and ##STR21## wherein R.sub.2, R.sub.3 and R.sub.4 are --CH.sub.2 CO.sub.2 H.